Methods and compositions for separating rare cells from fluid samples

ABSTRACT

The present invention includes methods of enriching rare cells, such as cancer cells, from biological samples, such as blood samples. The methods include performing at least one debulking step on a blood sample and selectively removing at least one type undesirable component from the blood sample to obtain a blood sample that is enriched in a rare cell of interest. In some embodiments magnetic beads coupled to specific binding members are used to selectively removed components.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/497,919 entitled, “Methods and Compositions for Separating Rare Cellsfrom Fluid Samples,” filed Aug. 2, 2006, now pending, the disclosure ofwhich is herein incorporated by reference in its entirety. U.S. patentapplication Ser. No. 11/497,919 claims benefit of priority to U.S.Patent Application Ser. No. 60/704,601 entitled, “Improved Methods andCompositions for Separating Rare Cells From Fluid Samples,” filed Aug.2, 2005, now expired, and is herein incorporated by reference in itsentirety. U.S. patent application Ser. No. 11/497,919 is also acontinuation-in-part of U.S. patent application Ser. No. 10/701,684,entitled “Methods, Compositions, and Automated Systems for SeparatingRare Cells from Fluid Samples” filed Nov. 4, 2003, now patented as U.S.Pat. No. 7,166,443, which is a continuation-in-part of U.S. patentapplication Ser. No. 10/268,312, entitled “Methods, Compositions, andAutomated Systems for Separating Rare Cells from Fluid Samples” filedOct. 10, 2002, now patented as U.S. Pat. No. 6,949,355, which claimsbenefit of priority to U.S. Provisional Patent application Ser. No.60/348,228, filed on Oct. 29, 2001, now expired, entitled “Methods andautomated systems for separating rare cells from fluid samples” and U.S.Provisional Patent application No. 60/328,724, filed Oct. 11, 2001, nowexpired, entitled “Methods and automated systems for separating rarecells from fluid samples”, and U.S. Provisional Patent application No.60/394,517, filed on Jul. 9, 2002, now expired, entitled “Methods andautomated systems for separating rare cells from fluid samples”, all ofwhich are incorporated by reference herein in their entirety.

BACKGROUND OF THE INVENTION

The present invention relates generally to the field of bioseparation,and in particular to the field of biological sample processing.

Sample preparation is a necessary step for many genetic, biochemical,and biological analyses of biological and environmental samples. Samplepreparation frequently requires the separation of sample components ofinterest from the remaining components of the sample. Such separationsare often labor intensive and difficult to automate.

In many cases it is necessary to analyze relatively rare components of asample. In this case, it may be necessary both to increase theconcentration of the rare components to be analyzed, and to removeundesirable components of the sample that can interfere with theanalysis of the components of interest. Thus, a sample must be“debulked” to reduce its volume, and in addition subjected to separationtechniques that can enrich the components of interest. This isparticularly true of biological samples, such as ascites fluid, lymphfluid, or blood, that can be harvested in large amounts, but that cancontain minute percentages of target cells (such as virus-infectedcells, anti-tumor T-cells, inflammatory cells, cancer cells, or fetalcells) whose separation is of critical importance for understanding thebasis of disease states as well as for diagnosis and development oftherapies.

Filtration has been used as a method of reducing the volume of samplesand separating sample components based on their ability to flow throughor be retained by the filter. Typically membrane filters are used insuch applications in which the membrane filters have interconnected,fiber-like, structure distribution and the pores in the membrane are notdiscretely isolated; instead the pores are of irregular shapes and areconnected to each other within the membrane. The so-called “pore” sizereally depends on the random tortuosity of the fluid-flow patches (e.g.pores) in the membrane. While the membrane filters can be used for anumber of separation applications, the variation in the pore size andthe irregular shapes of the pores prevent them being used for precisefiltration based on particle size and other properties.

Microfabricated filters have been made for certain cellular or molecularseparation. These microfabricated structures do not have pores, butrather include channels that are microetched into one or more chips, byusing “bricks” (see, for example, U.S. Pat. No. 5,837,115 issued Nov.17, 1998 to Austin et al., incorporated by reference) or dams see, forexample, U.S. Pat. No. 5,726,026 issued Mar. 10, 1998 to Wilding et al.,incorporated by reference) that are built onto the surface of a chip.While these microfabricated filters have precise geometries, theirlimitations are that the filtration area of the filter is small, limitedby the geometries of these filters, so that these filters can processonly small volumes of the fluid sample.

Blood samples provide special challenges for sample preparation andanalysis. Blood samples are easily obtained from subjects, and canprovide a wealth of metabolic, diagnostic, prognostic, and geneticinformation. However, the great abundance of non-nucleated red bloodcells, and their major component hemoglobin, can be an impediment togenetic, metabolic, and diagnostic tests. The debulking of red bloodcells from peripheral blood has been accomplished using different layersof dense solutions (for example, see U.S. Pat. No. 5,437,987 issued Aug.1, 1995 to Teng, Nelson N. H. et al). Long chain polymers such asdextran have been used to induce the aggregation of red blood cellsresulting in the formation of long red blood cell chains (Sewchand L S,Canham P B. (1979) ‘Modes of rouleaux formation of human red blood cellsin polyvinylpyrrolidone and dextran solutions’ Can. J. Physiol.Pharmacol. 57(11):1213-22. However, the efficiency of these solutions inremoving red blood cells is less than optimal, especially where theseparation or enrichment of rare cells, such as, for example, fetalcells from maternal blood or cancer cells from a patient, is desirable.

Exfoliated cells in body fluids (e.g. sputum, urine, or even asceticfluid or other effusions) present a significant opportunity fordetection of precancerous lesions and for eradication of cancer at earlystages of neoplastic development. For example, urine cytology isuniversally accepted as the noninvasive test for the diagnosis andsurveillance of transitional cell carcinoma (Larsson et al (2001)Molecular Diagnosis 6: 181-188). However, in many cases, the cytologicidentification of abnormal exfoliated cells has been limited by thenumber of abnormal cells isolated. For routine urine cytology (Ahrendtet al. (1999) J. Natl. Cancer Inst. 91: 299-301), the overallsensitivity is less than 50%, which varies with tumor grade, tumorstage, and urine collection and processing methods used. Molecularanalysis (e.g. using in situ hybridization, PCR, microarrays, etc) ofabnormal exfoliated cells in body fluids based on molecular and geneticbiomarkers can significantly improve the cytology sensitivity. Bothbiomarker studies and use of biomarkers for clinical practice wouldrequire a relative pure exfoliated cell population enriched from bodyfluids comprising not only exfoliated cells but also normal cells,bacteria, body fluids, body proteins and other cell debris. Thus, thereis an immediate need for developing an effective enrichment method forenriching and isolating exfoliated abnormal cells from body fluids.

Current approaches for enriching and preparing exfoliated cells frombody fluids are through media based separation, antibody capture,centrifugation and membrane filtration. While these techniques aresimple and straightforward, they suffer from a number of limitations,including: inadequate efficiency for rare cell enrichment; lowsensitivity of rare cell detection; difficulty in handling large volumesamples; inconsistency of the enrichment performance; andlabor-intensiveness of separation procedure.

There is a need to provide methods of sample preparation that areefficient and automatable that can process relatively large samplevolumes, such as large volumes of biological fluid samples, and separatetarget cells. The present invention provides these and other benefits.

BRIEF SUMMARY OF THE INVENTION

The present invention recognizes that diagnosis, prognosis, andtreatment of many conditions can depend on the enrichment of rare cellsfrom a complex fluid sample. Often, enrichment can be accomplished byone or more separation steps. In particular, the separation of fetalcells from maternal blood samples, can greatly aid in the detection offetal abnormalities or a variety of genetic conditions. In addition, thepresent invention recognizes that the enrichment or separation of raremalignant cells from patient samples, such as the isolation of cancerouscells from patient body fluid samples, can aid in the detection andtyping of such malignant cells and therefore aid in diagnosis andprognosis, as well as in the development of therapeutic modalities forpatients.

A first aspect of the present invention are methods of enriching rarecells from a peripheral blood sample in which during washing of a bloodsample, the blood sample is centrifuged at a speed that enhances therecovery of a rare cell type of interest. In one embodiment of thisaspect, a blood sample (obtained from a pregnant female) is washed bycentrifuging at a speed that enhances recovery of mononucleated fetalcells. In another embodiment of this aspect, a maternal blood sample iswashed by centrifuging at a speed that enhances recovery of fetalpolynucleated or less healthy fetal cells (e.g. trophoblasts, apoptoticor dying cells).

In a related aspect of the present invention, methods of enriching rarecells from a maternal blood sample are provided in which recovery of arare cell type of interest is enhanced by use of a maternal blood sampleof a particular gestational age or window. In one embodiment of thisaspect, a maternal blood sample of a particular gestational age orwindow is harvested for enrichment of nucleated fetal cells. In anotherembodiment of this aspect, a maternal blood sample of a particulargestational age or window is harvested for enrichment of fetaltrophoblasts or nucleated red blood cells.

A third aspect of the present invention are methods of isolating rarefetal cells from a maternal blood sample comprising: providing amaternal blood sample; washing the maternal supernatant twice bycentrifugation, in which after the second wash centrifugation a secondwash supernatant and a second wash pellet are obtained; resuspending thesecond wash pellet to obtain second wash pellet cells; resuspending thesecond wash pellet cells; centrifuging the second wash supernatant toobtain pelleted second wash supernatant cells; resuspending the pelletedsecond wash supernatant cells and adding the second wash supernatantcells to the resuspended second wash pellet cells to obtain combinedsecond wash pellet and second wash supernatant cells; and enriching rarecells from the combined second wash pellet and second wash supernatantcells. The enriching procedure can include any combination of: debulkingthe sample, removing one or more undesirable components from the sample,and separating one or more desirable components of the sample.

A fourth aspect of the present invention is the use of an antibody ormolecule that specifically binds a platelet surface molecule or moietyor serum protein(s) with lesser binding to the desired cells.Nonlimiting examples include an antibody or molecule that binds CD31,CD36, CD41, CD42 (a,b,c), CD51 and CD51/61. The antibody may be utilizedto remove platelets from a blood sample. The antibody can optionally bebound to a solid support. In preferred embodiments of the presentinvention, the antibody can be used to remove platelets from a bloodsample in a procedure for enriching rare cells from a blood sample.

A fifth aspect of the present invention is a magnet configuration forefficient separation of magnetic particles from a sample. In preferredembodiments, the magnet configuration comprises multiple magnetspositioned around a vessel such as a tube. The present invention alsoincludes methods of separating sample components from a fluid sampleusing magnetic particles and a magnet configuration comprising multiplemagnets positioned around the sample vessel.

A sixth aspect of the present invention is a method of making amicrofabricated filter that comprises at least one pore for filtering afluid sample. The method may include providing a chip, depositing adielectric layer along opposing surfaces of a portion of the chip,removing at least one region of the dielectric layer and at least oneregion of the chip that is to become at least one pore and removing theremaining dielectric layer. The method may also include forming a cavityprior to forming the at least one pore and forming the at least one porein substantial alignment with cavity. The present invention alsoincludes filters made using the methods of the present invention.

The present invention also comprises methods of treating or modifying(e.g. chemically) a filter of the present invention to increase theefficiency of filtering a fluid sample, such as a fluid sample thatcomprises cells. The present invention also includes filters treatedusing the methods of the present invention.

A seventh aspect of the present invention is a method for enriching rarecells from a blood sample that comprises: debulking the blood sample;specifically labeling at least one component of the blood sample with adetectable label; and separating one or more components of the sampleutilizing components other than the at least one component of the samplethat is labeled with a detectable label. The one or more components ofthe blood sample labeled with a detectable label are preferablydesirable components of the sample, but can also be undesirablecomponents of the sample, or both desirable and undesirable componentsof the sample.

In some preferred embodiments, a blood sample is debulked by adding asolution that selectively sediments red blood cells, and a labelingreagent that labels desirable cells is added to the blood sample at thesame time as the debulking solution. Preferably, specific bindingmembers that bind undesirable sample components are also added with thedebulking solution.

In some preferred embodiments, after debulking and the removal ofundesirable components from the blood sample, labeled desirable cellsare further enriched or isolated using fluorescence activated cellsorting or laser cytometry. In some preferred embodiments, afterdebulking and the removal of undesirable components from the bloodsample, labeled desirable cells are further analyzed using spectralimaging, fluorescence microscopy, visible light microscopy, or manual orautomated image analysis.

An eighth aspect of the present invention is a method of enriching arare cell, such as a cancer cell, from a biological sample includingperforming at least one debulking step on a blood sample and selectivelyremoving at least one type of undesirable component from the bloodsample to enrich a rare cell of interest in the blood sample. Selectiveremoval may occur by contacting the blood sample with one or morespecific binding members that are specific to one or more types ofundesirable components, the specific binding members being optionallycoupled to a solid support such as a microbead or magnetic bead.Examples of suitable specific binding members are antibodies or antibodyfragments such as those capable of binding CD3, CD11b, CD14, CD17, CD31,CD36, CD41, CD42 (a,b,c), CD45, CD50, CD51, CD51/61, CD53, CD63, CD69,CD81, CD84, CD102 or CD166. White blood cells are one type ofundesirable component. Debulking, such as removing red blood cells, maybe performed using nonlimiting methods such as sedimentation, lysis,centrifugation and the like. Rare cells, such a cancer cells or othernon-hematopoietic cells may be labeled and/or further isolated.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is the top view of a region of a microfabricated chip of thepresent invention. The dark areas are the precision manufactured slotsin the filter that has a filtration area of 1 cm².

FIG. 2 is a schematic representation of a microfabricated filter of thepresent invention.

FIG. 2A) the top view, showing an 18×18 mm² microfabricated filterhaving a filtration area (1) of 10×10 mm². FIG. 2B) an enlargement of asection of the top view, showing the slots (2) having dimensions of 4microns×50 microns, with the center to center distance between slots of12 microns, and their parallel alignment. FIG. 2C) a cross-sectionalview of the microfabricated filter, with the slots extending through thefilter substrate.

FIG. 3A and FIG. 3B depict filters of the present invention havingelectrodes incorporated into their surfaces. FIG. 3A: a 20-foldmagnification of a portion of a microfabricated filter having 2 micronslot widths. FIG. 3B: a 20-fold magnification of a portion of amicrofabricated filter having 3 micron slot widths.

FIG. 4 depicts a cross section of a pore in a microfabricated filter ofthe present invention. The pore depth corresponds to the filterthickness. Y represents the right angle between the surface of thefilter and the side of a pore cut perpendicularly through the filter,while X is the tapering angle by which a tapered pore differs in itsdirection or orientation through the filter from a nontapered pore.

FIG. 5 depicts a filtration unit of the present invention having amicrofabricated filter (3) separating the filtration chamber into anupper antechamber (4) and a post-filtration subchamber (5). The unit hasvalves to control fluid flow into and out of the unit: valve A (6)controls the flow of sample from the loading reservoir (10) into thefiltration unit, valve B (7) controls fluid flow through the chamber byconnection to a syringe pump, and valve C (8) is used for theintroduction of wash solution into the chamber.

FIG. 6 is a diagram of an automated system of the present invention thatcomprises an inlet for the addition of a blood sample (11); a filtrationchamber (12) that comprises acoustic mixing chips (13) andmicrofabricated filters (103); a magnetic capture column (14) havingadjacent magnets (15); a mixing/filtration chamber (112); a magneticseparation chamber (16) comprising an electromagnetic chip (17), and avessel for rare cell collection (18).

FIG. 7 depicts a three-dimensional perspective view of a filtrationchamber of the present invention that has two filters (203) thatcomprise slots (202) and a chip having acoustic elements (200) (theacoustic elements may not be visible on the chip surface, but are shownhere for illustrative purposes). In this simplified depiction, the widthof the slots is not shown.

FIG. 8 depicts a cross-sectional view of a filtration chamber of thepresent invention having two filters (303) after filtering has beencompleted, and after the addition of magnetic beads (19) to a samplecomprising target cells (20). The acoustic elements are turned on duringa mixing operation.

FIG. 9 depicts a cross-sectional view of a feature of an automatedsystem of the present invention: a magnetic capture column (114).Magnets (115) are portioned adjacent to the separation column.

FIG. 10 depicts a three-dimensional perspective view of a chamber (416)of an automated system of the present invention that comprises amultiple force chip that can separate rare cells from a fluid sample.The chamber has an inlet (429) and an outlet (430) for fluid flowthrough the chamber. A cut-away view shows the chip has an electrodelayer (427) that comprises an electrode array for dielectrophoreticseparation and an electromagnetic layer (417) that compriseselectromagnetic units (421) an electrode array on another layer. Targetcells (420) are bound to magnetic beads (419) for electromagneticcapture.

FIG. 11 shows a graph illustrating the theoretical comparison betweenthe DEP spectra for an nRBC (Xs) and a RBC (circles) when the cells aresuspended in a medium of electrical conductivity of 0.2 S/m.

FIG. 12 shows FISH analysis of nucleated fetal cells isolated using themethods of the present invention using a Y chromosome marker that hasdetected a male fetal cell in a maternal blood sample.

FIG. 13 shows a process flow chart for enriching fetal nucleated RBCsfrom maternal blood.

FIG. 14 is a schematic depiction of a filtration unit of the presentinvention.

FIG. 15 shows a model of an automated system of the present invention.

FIGS. 16A-M depict the filtration process of an automated system of thepresent invention. FIG. 16A) shows the filtration unit having a loadingreservoir (510) connected through a valve (506) to a filtration chamberthat comprises an antechamber (504) separated from a post-filtrationsubchamber (505) by a microfabricated filter (503). A wash pump (526) isconnected to the lower chamber through a valve (508) for pumping washbuffer (524) through the lower subchamber. Another valve (507) leads toanother negative pressure pump used to promote fluid flow through thefiltration chamber and out through an exit conduit (530). A collectionvessel (518) can reversibly engage the upper chamber (504). FIG. 16B)shows a blood sample (525) loaded into the loading reservoir (510). InFIG. 16C) the valve (507) that leads to a negative pressure pump used topromote fluid flow through the filtration chamber is open, and FIG. 16D)and FIG. 16E) show the blood sample being filtered through the chamber.In FIG. 16F) wash buffer introduced through the loading reservoir isfiltered through the chamber. In FIG. 16G), valve (508) is open, whilethe loading reservoir valve (506) is closed, and wash buffer is pumpedfrom the wash pump (526) into the lower chamber. In FIG. 16H) thefiltration valve (507) and wash pump valve (508) are closed and in FIG.16I) and FIG. 16J) the chamber is rotated 90 degrees. FIG. 16K) showsthe collection vessel (518) engaging the antechamber (504) so that fluidflow generated by the wash pump (526) causes rare target cells (520)retained in the antechamber to flow into the collection tube.

FIG. 17A and FIG. 17B depict a fluorescently labeled breast cancer cellin a background of unlabeled blood cells after enrichment bymicrofiltration. FIG. 17A) phase contrast microscopy of filtered bloodsample. FIG. 17B) fluorescence microscopy of the same field shown inFIG. 17A.

FIG. 18A and FIG. 18B depict two configurations of dielectrophoresischips of the present invention. FIG. 18A) chip with interdigitatedelectrode geometry; FIG. 18B) chip with castellated electrode geometry.

FIG. 19A and FIG. 19B depict a separation chamber of the presentinvention comprising a dielectrophoresis chip. FIG. 19A) Cross-sectionalview of the chamber, FIG. 19B) top view showing the chip.

FIG. 20 is a graph illustrating the theoretical comparison between theDEP spectra for MDA231 cancer cells (solid line) T-lymphocytes (dashedline) and erythrocytes (small dashes) when the cells are suspended in amedium of electrical conductivity of 10 mS/m.

FIG. 21A and FIG. 21B depict breast cancer cells from a spiked bloodsample retained on electrodes of a dielectrophoresis chip. FIG. 21Ashows phase contrast microscopy of the spiked blood sample on thedielectrophoresis chip. FIG. 21B shows fluorescence microscopy offluorescently labeled breast cancer cells in the same field shown inFIG. 21A.

FIG. 22 depicts white blood cells of a blood sample retained onelectrodes of a dielectrophoresis chip.

FIG. 23 is a schematic representation of a filtration unit of anautomated system of the present invention. The filtration unit has aloading reservoir (610) connected through valve A (606) to a filtrationchamber that comprises an antechamber (604) separated from apost-filtration subchamber (605) by a microfabricated filter (603). Asuction-type pump can be attached through tubing that connects to thewaste port (634), where filtered sample exits the chamber. A side port(632) can be used for attaching a syringe pump for pumping wash bufferthrough the lower subchamber (605). After the filtration process, thefiltration chamber (including the antechamber (604), post-filtrationsubchamber (605), filter (603), and side port (632), all depicted withinthe circle in the figure) can rotate within the frame (636) of thefiltration unit, so that enriched cells of the antechamber can becollected via the collection port (635).

FIG. 24 is a diagram showing the overall process of fetal cellenrichment from a blood sample, and the presence of enriched fetal cellsin the supernatant of a second wash of the blood sample (box labeledSupernatant (W2)) and in the retained cells after the filtration step(box labeled Enriched cells). The diagram shows, from upper left tolower right, blood cell processing steps” two washes (W1 and W2),Selective sedimentation of red blood cells and removal of white bloodcells with a combined reagent (AVIPrep+AVIBeads+Antibodies), Filtrationof the supernatant of the sedimentation, and collection of enrichedfetal cells. The diagram shows the level of enrichment of nucleatedcells of various sample fractions during the procedure, and the samplefractions that were analyzed using FISH.

FIG. 25 is a photograph of a slide containing a control (FIG. 25A) and aplatelet depleted blood sample (FIG. 25B). The slides were stained witha Benzidine Wright-Giemsa staining protocol. FIG. 25A shows a controlsample and FIG. 25B shows a sample treated with a biotinylated CD31antibody used in combination with neutravidin coated magnetic beeds.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Generally, the nomenclatureused herein and the manufacture procedures for devices and components aswell as the laboratory procedures described below are well known andcommonly employed in the art. Conventional methods are used for theseprocedures, such as those provided in the art and various generalreferences. Where a term is provided in the singular, the inventors alsocontemplate the plural of that term. As employed throughout thedisclosure, the following terms, unless otherwise indicated, shall beunderstood to have the following meanings:

A “component” of a sample or “sample component” is any constituent of asample, and can be an ion, molecule, compound, molecular complex,organelle, virus, cell, aggregate, or particle of any type, includingcolloids, aggregates, particulates, crystals, minerals, etc. A componentof a sample can be soluble or insoluble in the sample media or aprovided sample buffer or sample solution. A component of a sample canbe in gaseous, liquid, or solid form. A component of a sample may be amoiety or may not be a moiety.

A “moiety” or “moiety of interest” is any entity whose manipulation isdesirable. A moiety can be a solid, including a suspended solid, or canbe in soluble form. A moiety can be a molecule. Molecules that can bemanipulated include, but are not limited to, inorganic molecules,including ions and inorganic compounds, or can be organic molecules,including amino acids, peptides, proteins, glycoproteins, lipoproteins,glycolipoproteins, lipids, fats, sterols, sugars, carbohydrates, nucleicacid molecules, small organic molecules, or complex organic molecules. Amoiety can also be a molecular complex, can be an organelle, can be oneor more cells, including prokaryotic and eukaryotic cells, or can be oneor more etiological agents, including viruses, parasites, or prions, orportions thereof. A moiety can also be a crystal, mineral, colloid,fragment, mycelle, droplet, bubble, or the like, and can comprise one ormore inorganic materials such as polymeric materials, metals, minerals,glass, ceramics, and the like. Moieties can also be aggregates ofmolecules, complexes, cells, organelles, viruses, etiological agents,crystals, colloids, or fragments. Cells can be any cells, includingprokaryotic and eukaryotic cells. Eukaryotic cells can be of any type.Of particular interest are cells such as, but not limited to, whiteblood cells, malignant cells, stem cells, progenitor cells, fetal cells,and cells infected with an etiological agent, and bacterial cells.Moieties can also be artificial particles such polystyrene microbeads,microbeads of other polymer compositions, magnetic microbeads, andcarbon microbeads.

As used herein, “manipulation” refers to moving or processing of themoieties, which results in one-, two- or three-dimensional movement ofthe moiety, whether within a single chamber or on a single chip, orbetween or among multiple chips and/or chambers. Moieties that aremanipulated by the methods of the present invention can optionally becoupled to binding partners, such as microparticles or microbeads.Non-limiting examples of the manipulations include transportation,capture, focusing, enrichment, concentration, aggregation, trapping,repulsion, levitation, separation, isolation or linear or other directedmotion of the moieties, detection, identification, characterization,culturing and the like. For effective manipulation of moieties coupledto binding partners, the binding partner and the physical force used inthe method must be compatible. For example, binding partners withmagnetic properties may be used with magnetic force. Thus, magneticmicrobeads may be used in a magnetic field to manipulate a moiety.Similarly, binding partners with certain dielectric properties, e.g.,plastic particles, polystyrene microbeads, must be used withdielectrophoretic force.

“Binding partner” refers to any substances that both bind to themoieties with desired affinity or specificity and are manipulatable withthe desired physical force(s). Non-limiting examples of the bindingpartners include cells, cellular organelles, viruses, microparticles oran aggregate or complex thereof, or an aggregate or complex ofmolecules.

A “microparticle” or “particle” is a structure of any shape and of anycomposition that is manipulatable by desired physical force(s). Themicroparticles used in the methods could have a dimension from about0.01 micron to about ten centimeters. Preferably, the microparticlesused in the methods have a dimension from about 0.1 micron to aboutseveral thousand microns. Such particles or microparticles can becomprised of any suitable material, such as glass or ceramics, and/orone or more polymers, such as, for example, nylon,polytetrafluoroethylene (TEFLON™), polystyrene, polyacrylamide,sepaharose, agarose, cellulose, cellulose derivatives, or dextran,and/or can comprise metals. Examples of microparticles include, but arenot limited to, plastic particles, ceramic particles, carbon particles,polystyrene microbeads, glass beads, magnetic beads, hollow glassspheres, metal particles, particles of complex compositions,microfabricated or micromachined particles, etc.

“Coupled” means bound. For example, a moiety can be coupled to amicroparticle by specific or nonspecific binding. As disclosed herein,the binding can be covalent or noncovalent, reversible or irreversible.

As used herein, “the moiety to be manipulated is substantially coupledonto surface of the binding partner” means that a percentage of themoiety to be manipulated is coupled onto surface of the binding partnerand can be manipulated by a suitable physical force via manipulation ofthe binding partner. Ordinarily, at least 0.1% of the moiety to bemanipulated is coupled onto surface of the binding partner. Preferably,at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of themoiety to be manipulated is coupled onto surface of the binding partner.

As used herein, “the moiety to be manipulated is completely coupled ontosurface of the binding partner” means that at least 90% of the moiety tobe manipulated is coupled onto surface of the binding partner.Preferably, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%of the moiety to be manipulated is coupled onto surface of the bindingpartner.

A “specific binding member” is one of two different molecules having anarea on the surface or in a cavity which specifically binds to and isthereby defined as complementary with a particular spatial and chemicalorganization of the other molecule. A specific binding member can be amember of an immunological pair such as antigen-antibody orantibody-antibody, can be biotin-avidin, biotin-streptavidin, orbiotin-neutravidin, ligand-receptor, nucleic acid duplexes, IgG-proteinA, DNA-DNA, DNA-RNA, RNA-RNA, and the like.

An “antibody” is an immunoglobulin molecule, and can be, as nonlimitingexample, an IgG, an IgM, or other type of immunoglobulin molecule. Asused herein, “antibody” also refers to a portion of an antibody moleculethat retains the binding specificity of the antibody from which it isderived (for example, single chain antibodies or Fab fragments).

A “nucleic acid molecule” is a polynucleotide. A nucleic acid moleculecan be DNA, RNA, or a combination of both. A nucleic acid molecule canalso include sugars other than ribose and deoxyribose incorporated intothe backbone, and thus can be other than DNA or RNA. A nucleic acid cancomprise nucleobases that are naturally occurring or that do not occurin nature, such as xanthine, derivatives of nucleobases, such as2-aminoadenine, and the like. A nucleic acid molecule of the presentinvention can have linkages other than phosphodiester linkages. Anucleic acid molecule of the present invention can be a peptide nucleicacid molecule, in which nucleobases are linked to a peptide backbone. Anucleic acid molecule can be of any length, and can be single-stranded,double-stranded, or triple-stranded, or any combination thereof.

“Homogeneous manipulation” refers to the manipulation of particles in amixture using physical forces, wherein all particles of the mixture havethe same response to the applied force.

“Selective manipulation” refers to the manipulation of particles usingphysical forces, in which different particles in a mixture havedifferent responses to the applied force.

A “fluid sample” is any fluid from which components are to be separatedor analyzed. A sample can be from any source, such as an organism, groupof organisms from the same or different species, from the environment,such as from a body of water or from the soil, or from a food source oran industrial source. A sample can be an unprocessed or a processedsample. A sample can be a gas, a liquid, or a semi-solid, and can be asolution or a suspension. A sample can be an extract, for example aliquid extract of a soil or food sample, an extract of a throat orgenital swab, or an extract of a fecal sample, or a wash of an internalarea of the body.

A “blood sample” as used herein can refer to a processed or unprocessedblood sample, i.e., it can be a centrifuged, filtered, extracted, orotherwise treated blood sample, including a blood sample to which one ormore reagents such as, but not limited to, anticoagulants or stabilizershave been added. An example of blood sample is a buffy coat that isobtained by processing human blood for enriching white blood cells.Another example of a blood sample is a blood sample that has been“washed” to remove serum components by centrifuging the sample to pelletcells, removing the serum supernatant, and resuspending the cells in asolution or buffer. Other blood samples include cord blood samples, bonemarrow aspirates, internal blood or peripheral blood. A blood sample canbe of any volume, and can be from any subject such as an animal orhuman. A preferred subject is a human.

A “rare cell” is a cell that is either 1) of a cell type that is lessthan 1% of the total nucleated cell population in a fluid sample, or 2)of a cell type that is present at less than one million cells permilliliter of fluid sample. A “rare cell of interest” is a cell whoseenrichment is desirable.

A “white blood cell” is a leukocyte, or a cell of the hematopoieticlineage that is not a reticulocyte or platelet and that can be found inthe blood of an animal or human. Leukocytes can include nature killercells (“NK cells”) and lymphocytes, such as B lymphocytes (“B cells”) orT lymphocytes (“T cells”). Leukocytes can also include phagocytic cells,such as monocytes, macrophages, and granulocytes, including basophils,eosinophils and neutrophils. Leukocytes can also comprise mast cells.

A “red blood cell” or “RBC” is an erythrocyte. Unless designated a“nucleated red blood cell” (“nRBC”) or “fetal nucleated red blood cell”or nucleated fetal red blood cell, as used herein, “red blood cell” isused to mean a non-nucleated red blood cell.

“Neoplastic cells” refers to abnormal cells that have uncontrolledcellular proliferation and can continue to grow after the stimuli thatinduced the new growth has been withdrawn. Neoplastic cells tend to showpartial or complete lack of structural organization and functionalcoordination with the normal tissue, and may be benign or malignant.

A “malignant cell” is a cell having the property of locally invasive anddestructive growth and metastasis. Examples of “malignant cells”include, but not limited to, leukemia cells, lymphoma cells, cancercells of solid tumors, metastatic solid tumor cells (e.g., breast cancercells, prostate cancer cells, lung cancer cells, colon cancer cells) invarious body fluids including blood, bone marrow, ascistic fluids,stool, urine, bronchial washes etc.

A “cancerous cell” is a cell that exhibits deregulated growth and, inmost cases, has lost at least one of its differentiated properties, suchas, but not limited to, characteristic morphology, non-migratorybehavior, cell-cell interaction and cell-signaling behavior, proteinexpression and secretion pattern, etc.

A “stem cell” is an undifferentiated cell that can give rise, throughone or more cell division cycles, to at least one differentiated celltype.

A “progenitor cell” is a committed but undifferentiated cell that cangive rise, through one or more cell division cycles, to at least onedifferentiated cell type. Typically, a stem cell gives rise to aprogenitor cell through one or more cell divisions in response to aparticular stimulus or set of stimuli, and a progenitor gives rise toone or more differentiated cell types in response to a particularstimulus or set of stimuli.

An “etiological agent” refers to any etiological agent, such as abacteria, fungus, protozoan, virus, parasite or prion that can infect asubject. An etiological agent can cause symptoms or a disease state inthe subject it infects. A human etiological agent is an etiologicalagent that can infect a human subject. Such human etiological agents maybe specific for humans, such as a specific human etiological agent, ormay infect a variety of species, such as a promiscuous human etiologicalagent.

“Subject” refers to any organism, such as an animal or a human. Ananimal can include any animal, such as a feral animal, a companionanimal such as a dog or cat, an agricultural animal such as a pig or acow, or a pleasure animal such as a horse.

A “chamber” is a structure that is capable of containing a fluid sample,in which at least one processing step can be performed. The chamber mayhave various dimensions and its volume may vary between ten microlitersand 0.5 liter.

A “filtration chamber” is a chamber through which or in which a fluidsample can be filtered.

A “filter” is a structure that comprises one or more pores or slots ofparticular dimensions (that can be within a particular range), thatallows the passage of some sample components but not others from oneside of the filter to the other, based on the size, shape, and/ordeformability of the particles. A filter can be made of any suitablematerial that prevents passage of insoluble particles, such as metal,ceramics, glass, silicon, plastics, polymers, fibers (such as paper orfabric), etc.

A “filtration unit” is a filtration chamber and the associated inlets,valves, and conduits that allow sample and solutions to be introducedinto the filtration chamber and sample components to be removed from thefiltration chamber. A filtration unit optionally also comprises aloading reservoir.

A “cartridge” is a structure that comprises at least one chamber that ispart of a manual or automated system and one or more conduits for thetransport of fluid into or out of at least one chamber. A cartridge mayor may not comprise one or more chips.

An “automated system for separating rare cells from a fluid sample” oran “automated system” is a device that comprises at least one filtrationchamber, automated means for directing fluid flow through the filtrationchamber, and at least one power source for providing fluid flow and,optionally, providing a signal source for the generation of forces onactive chips. An automated system of the present invention can alsooptionally include one or more active chips, separation chambers,separation columns, or permanent magnets.

A “port” is an opening in the housing of a chamber through which a fluidsample can enter or exit the chamber. A port can be of any dimensions,but preferably is of a shape and size that allows a sample to bedispensed into a chamber by pumping a fluid through a conduit, or bymeans of a pipette, syringe, or other means of dispensing ortransporting a sample.

An “inlet” is a point of entrance for sample, solutions, buffers, orreagents into a fluidic chamber. An inlet can be a port of a chamber, orcan be an opening in a conduit that leads, directly or indirectly, to achamber of an automated system.

An “outlet” is the opening at which sample, sample components, orreagents exit a fluidic chamber. The sample components and reagents thatleave a chamber can be waste, i.e., sample components that are not to beused further, or can be sample components or reagents to be recovered,such as, for example, reusable reagents or target cells to be furtheranalyzed or manipulated. An outlet can be a port of a chamber, butpreferably is an opening in a conduit that, directly or indirectly,leads from a chamber of an automated system.

A “conduit” is a means for fluid to be transported from a container to achamber of the present invention. Preferably a conduit directly orindirectly engages a port in the housing of a chamber. A conduit cancomprise any material that permits the passage of a fluid through it.Conduits can comprise tubing, such as, for example, rubber, Teflon, ortygon tubing. Conduits can also be molded out of a polymer or plastic,or drilled, etched, or machined into a metal, glass or ceramicsubstrate. Conduits can thus be integral to structures such as, forexample, a cartridge of the present invention. A conduit can be of anydimensions, but preferably ranges from 10 microns to 5 millimeters ininternal diameter. A conduit is preferably enclosed (other than fluidentry and exit points), or can be open at its upper surface, as acanal-type conduit.

A “chip” is a solid substrate on which one or more processes such asphysical, chemical, biochemical, biological or biophysical processes canbe carried out, or a solid substrate that comprises or supports one ormore applied force-generating elements for carrying out one or morephysical, chemical, biochemical, biological, or biophysical processes.Such processes can be assays, including biochemical, cellular, andchemical assays; separations, including separations mediated byelectrical, magnetic, physical, and chemical (including biochemical)forces or interactions; chemical reactions, enzymatic reactions, andbinding interactions, including captures. The micro structures ormicro-scale structures such as, channels and wells, bricks, dams,filters, electrode elements, electromagnetic elements, or acousticelements, may be incorporated into or fabricated on the substrate forfacilitating physical, biophysical, biological, biochemical, chemicalreactions or processes on the chip. The chip may be thin in onedimension and may have various shapes in other dimensions, for example,a rectangle, a circle, an ellipse, or other irregular shapes. The sizeof the major surface of chips of the present invention can varyconsiderably, e.g., from about 1 mm² to about 0.25 m². Preferably, thesize of the chips is from about 4 mm² to about 25 cm² with acharacteristic dimension from about 1 mm to about 5 cm. The chipsurfaces may be flat, or not flat. The chips with non-flat surfaces mayinclude channels or wells fabricated on the surfaces. A chip can haveone or more openings, such as pores or slots.

An “active chip” is a chip that comprises micro-scale structures thatare built into or onto a chip that when energized by an external powersource can generate at least one physical force that can perform aprocessing step or task or an analysis step or task, such as, but notlimited to, mixing, translocation, focusing, separation, concentration,capture, isolation, or enrichment. An active chip uses applied physicalforces to promote, enhance, or facilitate desired biochemical reactionsor processing steps or tasks or analysis steps or tasks. On an activechip, “applied physical forces” are physical forces that, when energy isprovided by a power source that is external to an active chip, aregenerated by micro-scale structures built into or onto a chip.

“Micro-scale structures” are structures integral to or attached on achip, wafer, or chamber that have characteristic dimensions of scale foruse in microfluidic applications ranging from about 0.1 micron to about20 mm. Example of micro-scale structures that can be on chips of thepresent invention are wells, channels, dams, bricks, filters, scaffolds,electrodes, electromagnetic units, acoustic elements, or microfabricatedpumps or valves. A variety of micro-scale structures are disclosed inU.S. patent application Ser. No. 09/679,024, having attorney docketnumber 471842000400, entitled “Apparatuses Containing Multiple ActiveForce Generating Elements and Uses Thereof” filed Oct. 4, 2000, hereinincorporated by reference in its entirety. Micro-scale structures thatcan, when energy, such as an electrical signal, is applied, generatephysical forces useful in the present invention, can be referred to as“physical force-generating elements” “physical force elements”, “activeforce elements”, or “active elements”.

A variety of micro-scale structures are disclosed in U.S. patentapplication Ser. No. 09/679,024, having attorney docket number471842000400, entitled “Apparatuses Containing Multiple Active ForceGenerating Elements and Uses Thereof” filed Oct. 4, 2000, hereinincorporated by reference in its entirety. Micro-scale structures thatcan, when energy, such as an electrical signal, is applied, generatephysical forces useful in the present invention, can be referred to as“physical force-generating elements” “physical force elements”, “activeforce elements”, or “active elements”.

A “multiple force chip” or “multiforce chip” is a chip that generatesphysical force fields and that has at least two different types ofbuilt-in structures each of which is, in combination with an externalpower source, capable of generating one type of physical field. A fulldescription of the multiple force chip is provided in U.S. applicationSer. No. 09/679,024 having attorney docket number 471842000400, entitled“Apparatuses Containing Multiple Active Force Generating Elements andUses Thereof” filed Oct. 4, 2000, herein incorporated by reference inits entirety.

“Acoustic forces” are the forces exerted, directly or indirectly onmoieties (e.g., particles and/or molecules) by an acoustic wave field.Acoustic forces can be used for manipulating (e.g., trapping, moving,directing, handling) particles in fluid. Acoustic waves, both standingacoustic wave and traveling acoustic wave, can exert forces directly onmoieties and such forces are called “acoustic radiation forces”.Acoustic wave may also exert forces on the fluid medium in which themoieties are placed, or suspended, or dissolved and result in so-calledacoustic streaming. The acoustic streaming, in turn, will exert forceson the moieties placed, suspended or dissolved in such a fluid medium.In this case, the acoustic wave fields can exert forces on moieties indirectly.

“Acoustic elements” are structures that can generate an acoustic wavefield in response to a power signal. Preferred acoustic elements arepiezoelectric transducers that can generate vibrational (mechanical)energy in response to applied AC voltages. The vibrational energy can betransferred to a fluid that is in proximity to the transducers, causingan acoustic force to be exerted on particles (such as, for example,cells) in the fluid. A description of acoustic forces and acousticelements can be found in U.S. patent application Ser. No. 09/636,104,filed Aug. 10, 2000, incorporated by reference in its entirety.

“Piezoelectic transducers” are structures capable of generating anacoustic field in response to an electrical signal. Non-limitingexamples of the piezoelectric transducers are ceramic disks (e.g. PZT,Lead Zirconium Titinate) covered on both surfaces with metal filmelectrodes, piezoelectric thin films (e.g. zinc-oxide).

“Mixing” as used herein means the use of physical forces to causeparticle movement in a sample, solution, or mixture, such thatcomponents of the sample, solution, or mixture become interspersed.Preferred methods of mixing for use in the present invention include useof acoustic forces.

“Processing” refers to the preparation of a sample for analysis, and cancomprise one or multiple steps or tasks. Generally a processing taskserves to separate components of a sample, concentrate components of asample, at least partially purify components of a sample, orstructurally alter components of a sample (for example, by lysis ordenaturation).

As used herein, “isolating” means separating a desirable samplecomponent from other nondesirable components of a sample, such thatpreferably, at least 15%, more preferably at least 30%, even morepreferably at least 50%, and further preferably, at least 80% of thedesirable sample components present in the original sample are retained,and preferably at least 50%, more preferably at least 80%, even morepreferably, at least 95%, and yet more preferably, at least 99%, of atleast one nondesirable component of the original component is removed,from the final preparation.

“Rare cells” are cells whose abundance in the original sample iseither 1) less than 1% of the total nucleated cell population in a fluidsample, or 2) present at less than one million cells per milliliter offluid sample.

“Enrich” means increase the concentration of a sample component of asample relative to other sample components (which can be the result ofreducing the concentration of other sample components), or increase theconcentration of a sample component. For example, as used herein,“enriching” nucleated fetal cells from a blood sample means increasingthe proportion of nucleated fetal cells to all cells in the bloodsample, enriching cancer cells of a blood sample can mean increasing theconcentration of cancer cells in the sample (for example, by reducingthe sample volume) or reducing the concentration of other cellularcomponents of the blood sample, and “enriching” cancer cells in a urinesample can mean increasing their concentration in the sample.

“Separation” is a process in which one or more components of a sampleare spatially separated from one or more other components of a sample. Aseparation can be performed such that one or more sample components ofinterest is translocated to or retained in one or more areas of aseparation apparatus and at least some of the remaining components aretranslocated away from the area or areas where the one or more samplecomponents of interest are translocated to and/or retained in, or inwhich one or more sample components is retained in one or more areas andat least some or the remaining components are removed from the area orareas. Alternatively, one or more components of a sample can betranslocated to and/or retained in one or more areas and one or moresample components can be removed from the area or areas. It is alsopossible to cause one or more sample components to be translocated toone or more areas and one or more sample components of interest or oneor more components of a sample to be translocated to one or more otherareas. Separations can be achieved through, for example, filtration, orthe use of physical, chemical, electrical, or magnetic forces.Nonlimiting examples of forces that can be used in separations aregravity, mass flow, dielectrophoretic forces, traveling-wavedielectrophoretic forces, and electromagnetic forces.

“Separating a sample component from a (fluid) sample” means separating asample component from other components of the original sample, or fromcomponents of the sample that are remaining after one or more processingsteps. “Removing a sample component from a (fluid) sample” meansremoving a sample component from other components of the originalsample, or from components of the sample that are remaining after one ormore processing steps.

“Capture” is a type of separation in which one or more moieties orsample components is retained in or on one or more areas of a surface,chamber, chip, tube, or any vessel that contains a sample, where theremainder of the sample can be removed from that area.

An “assay” is a test performed on a sample or a component of a sample.An assay can test for the presence of a component, the amount orconcentration of a component, the composition of a component, theactivity of a component, etc. Assays that can be performed inconjunction with the compositions and methods of the present inventioninclude, but not limited to, immunocytochemical assays, interphase FISH(fluorescence in situ hybridization), karyotyping, immunological assays,biochemical assays, binding assays, cellular assays, genetic assays,gene expression assays and protein expression assays.

A “binding assay” is an assay that tests for the presence orconcentration of an entity by detecting binding of the entity to aspecific binding member, or that tests the ability of an entity to bindanother entity, or tests the binding affinity of one entity for anotherentity. An entity can be an organic or inorganic molecule, a molecularcomplex that comprises, organic, inorganic, or a combination of organicand inorganic compounds, an organelle, a virus, or a cell. Bindingassays can use detectable labels or signal generating systems that giverise to detectable signals in the presence of the bound entity. Standardbinding assays include those that rely on nucleic acid hybridization todetect specific nucleic acid sequences, those that rely on antibodybinding to entities, and those that rely on ligands binding toreceptors.

A “biochemical assay” is an assay that tests for the presence,concentration, or activity of one or more components of a sample.

A “cellular assay” is an assay that tests for a cellular process, suchas, but not limited to, a metabolic activity, a catabolic activity, anion channel activity, an intracellular signaling activity, areceptor-linked signaling activity, a transcriptional activity, atranslational activity, or a secretory activity.

A “genetic assay” is an assay that tests for the presence or sequence ofa genetic element, where a genetic element can be any segment of a DNAor RNA molecule, including, but not limited to, a gene, a repetitiveelement, a transposable element, a regulatory element, a telomere, acentromere, or DNA or RNA of unknown function. As nonlimiting examples,genetic assays can be gene expression assays, PCR assays, karyotyping,or FISH. Genetic assays can use nucleic acid hybridization techniques,can comprise nucleic acid sequencing reactions, or can use one or moreenzymes such as polymerases, as, for example a genetic assay based onPCR. A genetic assay can use one or more detectable labels, such as, butnot limited to, fluorochromes, radioisotopes, or signal generatingsystems.

“FISH” or “fluorescence in situ hybridization” is an assay wherein agenetic marker can be localized to a chromosome by hybridization.Typically, to perform FISH, a nucleic acid probe that is fluorescentlylabeled is hybridized to interphase chromosomes that are prepared on aslide. The presence and location of a hybridizing probe can bevisualized by fluorescence microscopy. The probe can also include anenzyme and be used in conjunction with a fluorescent enzyme substrate.

“Karyotyping” refers to the analysis of chromosomes that includes thepresence and number of chromosomes of each type (for example, each ofthe 24 chromosomes of the human haplotype (chromosomes 1-22, X, and Y)),and the presence of morphological abnormalities in the chromosomes, suchas, for example, translocations or deletions. Karyotyping typicallyinvolves performing a chromosome spread of a cell in metaphase. Thechromosomes can then be visualized using, foe example, but not limitedto, stains or genetic probes to distinguish the specific chromosomes.

A “gene expression assay” (or “gene expression profiling assay”) is anassay that tests for the presence or quantity of one or more geneexpression products, i.e. messenger RNAs. The one or more types of mRNAscan be assayed simultaneously on cells of the interest from a sample.For different applications, the number and/or the types of mRNAmolecules to be assayed in the gene expression assays may be different.

A “protein expression assay” (or “protein expression profiling assay”)is an assay that tests for the presence or quantity of one or moreproteins. One or more types of protein can be assayed simultaneously onthe cells of the interest from a sample. For different applications, thenumber and/or the types of protein molecules to be assayed in theprotein expression assays may be different.

“Histological examination” refers to the examination of cells usinghistochemical or stains or specific binding members (generally coupledto detectable labels) that can determine the type of cell, theexpression of particular markers by the cell, or can reveal structuralfeatures of the cell (such as the nucleus, cytoskeleton, etc.) or thestate or function of a cell. In general, cells can be prepared on slidesand “stained” using dyes or specific binding members directly orindirectly bound to detectable labels, for histological examination.Examples of dyes that can be used in histological examination arenuclear stains, such as Hoescht stains, or cell viability stains, suchas Trypan blue, or cellular structure stains such as Wright or Giemsa,enzyme activity benzidine for HRP to form visible precipitate. Examplesof specific binding members that can be used in histological examinationof fetal red blood cells are antibodies that specifically recognizefetal or embryonic hemoglobin.

An “electrode” is a structure of highly electrically conductivematerial. A highly conductive material is a material with a conductivitygreater than that of surrounding structures or materials. Suitablehighly electrically conductive materials include metals, such as gold,chromium, platinum, aluminum, and the like, and can also includenonmetals, such as carbon and conductive polymers. An electrode can beany shape, such as rectangular, circular, castellated, etc. Electrodescan also comprise doped semi-conductors, where a semi-conductingmaterial is mixed with small amounts of other “impurity” materials. Forexample, phosphorous-doped silicon may be used as conductive materialsfor forming electrodes.

A “well” is a structure in a chip, with a lower surface surrounded on atleast two sides by one or more walls that extend from the lower surfaceof the well or channel. The walls can extend upward from the lowersurface of a well or channel at any angle or in any way. The walls canbe of an irregular conformation, that is, they may extend upward in asigmoidal or otherwise curved or multi-angled fashion. The lower surfaceof the well or channel can be at the same level as the upper surface ofa chip or higher than the upper surface of a chip, or lower than theupper surface of a chip, such that the well is a depression in thesurface of a chip. The sides or walls of a well or channel can comprisematerials other than those that make up the lower surface of a chip.

A “channel” is a structure in a chip with a lower surface and at leasttwo walls that extend upward from the lower surface of the channel, andin which the length of two opposite walls is greater than the distancebetween the two opposite walls. A channel therefore allows for flow of afluid along its internal length. A channel can be covered (a “tunnel”)or open.

A “pore” is an opening in a surface, such as a filter of the presentinvention, that provides fluid communication between one side of thesurface and the other. A pore can be of any size and of any shape, butpreferably a pore is of a size and shape that restricts passage of atleast one insoluble sample component from one side of a filter to theother side of a filter based on the size, shape, and deformability (orlack thereof), of the sample component.

A “slot” is an opening in a surface, such as a filter of the presentinvention. The slot length is longer than its width (slot length andslot width refer to the slots dimensions in the plane or the surface ofthe filter into which the sample components will go through, and slotdepth refers to the thickness of the filter). The term “slot” thereforedescribes the shape of a pore, which will in most cases be approximatelyrectangular, ellipsoid, or that of a quadrilateral or parallelogram.

“Bricks” are structures that can be built into or onto a surface thatcan restrict the passage of sample components between bricks. The designand use of one type of bricks (called “obstacles”) on a chip isdescribed in U.S. Pat. No. 5,837,115 issued Nov. 17, 1998 to Austin etal., herein incorporated by reference in its entirety.

A “dam” is a structure built onto the lower surface of a chamber thatextends upward toward the upper surface of a chamber leaving a space ofdefined width between the top of the dam and the top of the chamber.Preferably, the width of the space between the top of the dam and theupper wall of the chamber is such that fluid sample can pass through thespace, but at least one sample component is unable to pass through thespace based on its size, shape, or deformability (or lack thereof). Thedesign and use of one type of dam structure on a chip is described inU.S. Pat. No. 5,928,880 issued Jul. 27, 1999 to Wilding et al., hereinincorporated by reference in its entirety.

“Continuous flow” means that fluid is pumped or injected into a chamberof the present invention continuously during the separation process.This allows for components of a sample that are not selectively retainedin a chamber to be flushed out of the chamber during the separationprocess.

“Binding partner” refers to any substances that both bind to themoieties with desired affinity or specificity and are manipulatable withthe desired physical force(s). Non-limiting examples of the bindingpartners include microparticles.

A “microparticle” is a structure of any shape and of any compositionthat is manipulatable by desired physical force(s). The microparticlesused in the methods could have a dimension from about 0.01 micron toabout ten centimeters. Preferably, the microparticles used in themethods have a dimension from about 0.1 micron to about several hundredmicrons. Such particles or microparticles can be comprised of anysuitable material, such as glass or ceramics, and/or one or morepolymers, such as, for example, nylon, polytetrafluoroethylene(TEFLON™), polystyrene, polyacrylamide, sepaharose, agarose, cellulose,cellulose derivatives, or dextran, and/or can comprise metals. Examplesof microparticles include, but are not limited to, magnetic beads,magnetic particles, plastic particles, ceramic particles, carbonparticles, polystyrene microbeads, glass beads, hollow glass spheres,metal particles, particles of complex compositions, microfabricatedfree-standing microstructures, etc. The examples of microfabricatedfree-standing microstructures may include those described in “Design ofasynchronous dielectric micromotors” by Hagedorn et al., in Journal ofElectrostatics, Volume: 33, Pages 159-185 (1994). Particles of complexcompositions refer to the particles that comprise or consists ofmultiple compositional elements, for example, a metallic sphere coveredwith a thin layer of non-conducting polymer film.

“A preparation of microparticles” is a composition that comprisesmicroparticles of one or more types and can optionally include at leastone other compound, molecule, structure, solution, reagent, particle, orchemical entity. For example, a preparation of microparticles can be asuspension of microparticles in a buffer, and can optionally includespecific binding members, enzymes, inert particles, surfactants,ligands, detergents, etc.

Other technical terms used herein have their ordinary meaning in the artthat they are used, as exemplified by a variety of technicaldictionaries.

Introduction

The present invention recognizes that analysis of complex fluids, suchas biological fluid samples, can be confounded by many sample componentsthat can interfere with the analysis. Sample analysis can be even moreproblematic when the target of the analysis is a rare cell type, forexample, when the target cells are fetal cells present in maternal bloodor malignant cells present in the blood or urine of a patient. Inprocessing such samples, it is often necessary to both “debulk” thesample, by reducing the volume to a manageable level, and to enrich thepopulation of rare cells that are the target of analysis. Procedures forthe processing of fluid samples are often time consuming andinefficient. The present invention provides efficient methods andautomated systems for the enrichment of rare cells from fluid samples.

As a non-limiting introduction to the breath of the present invention,the present invention includes several general and useful aspects,including:

1) a microfabricated filter for filtering a fluid sample. Amicrofabricated filter of the present invention comprises at least onetapered pore, and preferably comprises at least two tapered pores whosevariation in size is 20% or less.

2) a method of enriching rare cells of a fluid sample using amicrofabricated filter of the present invention.

3) solutions for the selective sedimentation of red blood cells (RBCs)from a blood sample comprising a red blood cell aggregating agent and atleast one specific binding member that selectively binds RBCs. Thesolution could also comprise at least one specific binding member thatselectively binds other undesired components from a blood sample.Solutions of the present invention include a combined solution for rarecell enrichment that comprise dextran, at least one specific bindingmember that selectively binds RBCs, and at least one additional specificbinding member for the removal of undesirable sample components otherthan RBCs such as but not limiting to the following examples, e.g. WBCs,platelets or serum proteins).

4) methods of using selective RBC sedimentation solutions and combinedsolutions for enriching rare cells of a fluid sample.

5) an automated system for processing a fluid sample that includes: atleast one filtration chamber that comprises or engages one or moremicrofabricated filters of the present invention; automated means fordirecting fluid flow through the one or more filtration chambers of theautomated system, and means for collecting enriched rare cells.

6) a method of using an automated system for separating rare cells froma fluid sample that includes: introducing a fluid sample into anautomated system of the present invention, filtering the fluid sampleusing at least one filtration chamber of the automated system; andcollecting enriched rare cells from at least one outlet or at least onevessel of the automated system. Preferably, the method also includesremoving undesirable components of the fluid sample or separating rarecells of the sample in at least one vessel, chamber, or column of thepresent invention. A preferred fluid sample is an effusion, blood, orurine sample, and rare cells that can be enriched from such sampleinclude nucleated fetal cells, stem cells and cancer cells.

7) an automated system for processing a fluid sample that includes:automated fluid volume sensing means for sensing the volume of at leastone sample or a portion thereof provided in a tube or vessel; at leastone filtration chamber that comprises or engages one or moremicrofabricated filters of the present invention; automated means fordirecting fluid flow through the one or more filtration chambers of theautomated system, and means for collecting enriched rare cells.

8) a method of using an automated system for separating rare cells froma fluid sample that includes: providing a fluid sample in a tube orvessel; using automated fluid sensing means of the automated system todetermine the volume of the sample, or a portion thereof; filtering thefluid sample using at least one filtration chamber of the automatedsystem; and collecting enriched rare cells from at least one outlet orat least one vessel of the automated system. Preferably, the method alsoincludes removing undesirable components of the fluid sample orseparating rare cells of the sample in at least one vessel, chamber, orcolumn of the present invention. A preferred fluid sample is aneffusion, blood, or urine sample, and rare cells that can be enrichedfrom such sample include nucleated fetal cells, stem cells, and cancercells.

9) a method of enriching a rare cell, such as a cancer cell, from abiological sample including performing at least one debulking step on ablood sample and selectively removing at least one type of undesirablecomponent from the blood sample to enrich a rare cell of interest in theblood sample. Selective removal may occur by contacting the blood samplewith one or more specific binding members that are specific to one ormore types of undesirable components, the specific binding members beingoptionally coupled to a solid support such as a microbead or magneticbead. Examples of suitable specific binding members are antibodies orantibody fragments such as those capable of binding CD3, CD11b, CD14,CD17, CD31, CD36, CD41, CD42 (a,b,c), CD45, CD50, CD51, CD51/61, CD53,CD63, CD69, CD81, CD84, CD102 or CD166. White blood cells are one typeof undesirable component. Debulking, such as removing red blood cells,may be performed using nonlimiting methods such as sedimentation, lysis,centrifugation and the like. Rare cells, such a cancer cells or othernon-hematopietic cells may be labeled and/or further isolated.

These aspects of the invention, as well as others described herein, canbe achieved by using the methods, articles of manufacture andcompositions of matter described herein. To gain a full appreciation ofthe scope of the present invention, it will be further recognized thatvarious aspects of the present invention can be combined to makedesirable embodiments of the invention.

I. Microfabricated Filter

The present invention includes a microfabricated filter that comprisesat least one tapered pore, where a pore is an opening in the filter. Apore can be of any shape and any dimensions. For example, a pore can bequadrilateral, rectangular, ellipsoid, or circular in shape, or of othergeometric or non-geometric shape. A pore can have a diameter (or widestdimension) from about 0.1 micron to about 1000 microns, preferably fromabout 20 to about 200 microns, depending on the filtering application.Preferably, a pore is made during the machining of a filter, and ismicroetched or bored into the filter material that comprises a hard,fluid-impermeable material such as glass, silicon, ceramics, metal orhard plastic such as acrylic, polycarbonate, or polyimide. It is alsopossible to use a relatively nonhard surface for the filter that issupported on a hard solid support. Another aspect of this invention isto modify the material (for example but not limited to chemically orthermally modifying the material to silicon oxide or silicon nitride).Preferably, however, the filter comprises a hard material that is notdeformable by the pressure (such as suction pressure) used in generatingfluid flow through the filter.

A slot is a pore with a length that is greater than its width, where“length” and “width” are dimensions of the opening in the plane of thefilter. (The “depth” of the slot corresponds to the thickness of thefilter.) That is, “slot” describes the shape of the opening, which willin most cases be approximately rectangular or ellipsoid, but can alsoapproximate a quadrilateral or parallelogram. In preferred embodimentsof the present invention in which slot width is the critical dimensionin determining which sample components flow through or are retained bythe filter, the shape of the slot can vary at the ends (for example, beregular or irregular in shape, curved or angular), but preferably thelong sides of the slot are a consistent distance from one another formost of the length of the slot, that distance being the slot width. Thusthe long sides of a slot will be parallel or very nearly parallel, formost of the length of the slot.

Preferably, the filters used for filtration in the present invention aremicrofabricated or micromachined filters so that the pores or the slotswithin a filter can achieve precise and uniform dimensions. Such preciseand uniform pore or slot dimensions are a distinct advantage of themicrofabricated or micromachined filters of the present invention, incomparison with the conventional membrane filters made of materials suchas nylon, polycarbonate, polyester, mixed cellulose ester,polytetrafluoroethylene, polyethersulfone, etc. In the filters of thepresent invention, individual pores are isolated, have similar or almostidentical feature sizes, and are patterned on a filter. Such filtersallow precise separation of particles based on their sizes and otherproperties.

The filtration area of a filter is determined by the area of thesubstrate comprising the pores. The filtration area for microfabricatedfilters of the present invention can be between about 0.01 mm² and about0.1 m². Preferably, the filtration area is between about 0.25 mm² andabout 25 cm², and more preferably is between about 0.5 mm² and about 10cm². The large filtration areas allow the filters of the invention toprocess sample volumes from about 100 microliters to about 10 liters.The percent of the filtration area encompassed by pores can be fromabout 1% to about 70%, preferably is from about 10% to about 50%, andmore preferably is from about 15 to about 40%. The filtration area of amicrofabricated filter of the present invention can comprise any numberof pores, and preferably comprises at least two pores, but morepreferably the number of pores in the filtration area of a filter of thepresent invention ranges from about 4 to about 1,000,000, and even morepreferably ranges from about 100 to about 250,000. The thickness of thefilter in the filtration area can range from about 10 to about 500microns, but is preferably in the range of between about 40 and about100 microns.

The microfabricated filters of the present invention have slots or poresthat are etched through the filter substrate itself. The pores oropenings of the filters can be made by using microfabrication ormicromachining techniques on substrate materials, including, but notlimited to, silicon, silicon dioxide, ceramics, glass, polymers such aspolyimide, polyamide, etc. Various fabrication methods, as known tothose skilled in the art of microlithography and microfabrication (See,for example, Rai-Choudhury P. (Editor), Handbook of Microlithography,Micromachining and Microfabrication, Volume 2: Micromachining andmicrofabrication. SPIE Optical Engineering Press, Bellingham, Wash., USA(1997)), may be used. In many cases, standard microfabrication andmicromachining methods and protocols may be involved. One example ofsuitable fabrication methods is photolithography involving single ormultiple photomasks. The protocols in the microfabrication may includemany basic steps, for example, photolithographic mask generation,deposition of photoresist, deposition of “sacrificial” material layers,photoresist patterning with masks and developers, or “sacrificial”material layer patterning. Pores can be made by etching into thesubstrate under certain masking process so that the regions that havebeen masked are not etched off and the regions that have not beenmask-protected are etched off. The etching method can be dry-etchingsuch as deep RIE (reactive ion etching), laser ablation, or can be wetetching involving the use of wet chemicals.

Preferably, appropriate microfabrication or micromachining techniquesare chosen to achieve a desired aspect ratio for the filter pores. Theaspect ratio refers to the ratio of the slot depth (corresponding to thethickness of the filter in the region of the pores) to the slot width orslot length. The fabrication of filter slots with higher aspect ratios(i.e., greater slot depth) may involve deep etching methods. Manyfabrication methods, such as deep RIE, useful for the fabrication ofMEMS (micro electronic mechanical systems) devices can be used oremployed in making the microfabricated filters. The resulting pores can,as a result of the high aspect ratio and the etching method, have aslight tapering, such that their openings are narrower on one side ofthe filter than the other. For example, in FIG. 4, the angle Y, of ahypothetical pore bored straight through the filter substrate is 90degrees, and the tapering angle X by which a tapered pore of amicrofabricated filter of the present invention differs from theperpendicular is between about 0 degree and about 90 degrees, andpreferably between 0.1 degrees and 45 degrees and most preferablybetween about 0.5 degrees and 10 degrees, depending on the thickness ofthe filter (pore depth).

The present invention includes microfabricated filters comprising two ormore tapered pores. The substrate on which the filter pores, slots oropenings are fabricated or machined may be silicon, silicon dioxide,plastic, glass, ceramics or other solid materials. The solid materialsmay be porous or non-porous. Those who are skilled in microfabricationand micromachining fabrication may readily choose and determine thefabrication protocols and materials to be used for fabrication ofparticular filter geometries.

Using the microfabrication or micromachining methods, the filter slots,pores or openings can be made with precise geometries. Depending on thefabrication methods or materials used, the accuracy of a singledimension of the filter slots (e.g. slot length, slot width) can bewithin 20%, or less than 10%, or less than 5%. Thus, the accuracy of thecritical, single dimension of the filter pores (e.g. slot width foroblong or quadrilateral shaped slots) for the filters of the presentinvention are made within, preferably, less than 2 microns, morepreferably, less than 1 micron, or even more preferably less than 0.5micron.

Preferably, filters of the present invention can be made using thetrack-etch technique, in which filters made of glass, silicon, silicondioxides, or polymers such as polycarbonate or polyester with discretepores having relatively-uniform pore sizes are made. For example, thefilter can be made by adapting and applying the track-etch techniquedescribed at whatman.com/products/nucleopore/tech_frame.htm forNucleopore Track-etch membranes to filter substrates. In the techniqueused to make membrane filters, a thin polymer film is tracked withenergetic heavy ions to produce latent tracks on the film. The film isthen put in an etchant to produce pores.

Preferred filters for the cell separation methods and systems of thepresent invention include microfabricated or micromachined filters thatcan be made with precise geometries for the openings on the filters.Individual openings are isolated with similar or almost identicalfeature sizes and are patterned on a filter. The openings can be ofdifferent shapes such as, for example, circular, quadrilateral, orelliptical. Such filters allow precise separation of particles based ontheir sizes and other properties.

In a preferred embodiment of a microfabricated filter, individual poresare isolated and of a cylindrical shape, and the pore size is within a20% variation, where the pore size is calculated by the smallest andlargest dimension of the pore (width and length, respectively).

Filter Treatment or Modification

The present invention also includes methods of treating amicrofabricated filter to improve its filtering efficiency. In thesemethods, one or both surfaces of the filter is treated or coated ormodified to increase its filtering efficiency. In a preferred method,one or both surfaces of the filter is treated or modified to reduce thepossibility of sample components (such as but not limited to cells)interacting with or adhering to the filter.

A filter can be physically or chemically treated, for example, to alterits surface properties (e.g. hydrophobic, hydrophilic). For example, afilter can be heated or treated with oxygen plasma, modified to siliconnitride or can be treated with at least one acid or at least one base,to increase its hydrophilicity or surface charge. For example, a glassor silica filter can be heated to oxidize the surface of the filter.Heating times and temperatures can vary depending on the filter materialand the degree of oxidation desired. In one example, a glass filter canbe heated to a temperature of from about 200 to 1000 degrees Celsius forfrom about thirty minutes to twenty-four hours.

In another example, a filter can be treated with one or more acids orone or more bases to increase the hydrophilicity of the filter surface.In preferred embodiments, a filter that comprises glass or silica istreated with at least one acid.

An acid used in treating a filter of the present invention can be anyacid. As nonlimiting examples, the acid can be HCl, H₂SO₄, NaHSO₄, HSO₄,HNO₃, HF, H₃PO₄, HBr, HCOOH, or CH₃COOH. The acid can be of aconcentration about 0.1 N or greater, and preferably is about 0.5 N orhigher in concentration, and more preferably is greater than about 1 Nin concentration. For example, the concentration of acid preferably isfrom about 1 N to about 10 N. The incubation time can be from one minuteto days, but preferably is from about 5 minutes to about 2 hours.

Optimal concentrations and incubation times for treating amicrofabricated filter to increase its hydrophilicity can be determinedempirically. The microfabricated filter can be placed in a solution ofacid for any length of time, preferably for more than one minute, andmore preferably for more than about five minutes. Acid treatment can bedone under any non-freezing and non-boiling temperature, preferably at atemperature greater than or equal to room temperature.

Alternatively or in addition, a microfabricated filter of the presentinvention can be treated with a base, such as a basic solution, that cancomprise, as nonlimiting examples, NaOH, KOH, Ba(OH)₂, LiOH, CsOH, orCa(OH)₂. The basic solution can be of a concentration of about 0.01 N orgreater, and preferably is greater than about 0.05 N, and morepreferably greater than about 0.1 N in concentration. The ion transportmeasuring means can be placed in a solution of base for any length oftime, preferably for more than one minute, and more preferably for morethan about five minutes. Base treatment can be done under any non-frozenand non-boiling temperature, preferably at a temperature greater than orequal to room temperature.

The effectiveness of a physical or chemical treatment in increasing thehydrophilicity of a filter surface can be tested by measuring the spreadof a drop of water placed on the surface of a treated and non-treatedfilter, where increased spreading of a drop of uniform volume indicatesincreased hydrophilicity of a surface (FIG. 5). The effectiveness of afilter treatment can also be tested by incubating a treated filter withcells or biological samples to determine the degree of sample componentadhesion to the treated filter.

In another embodiment, the surface of a filter, such as but not limitedto a polymeric filter, can chemically treated to alter the surfaceproperties of the filter. For example, the surface of a glass, silica,or polymeric filter can be derivatized by any of various chemicaltreatments to add chemical groups that can decrease the interaction ofsample components with the filter surface.

One or more compounds can also be adsorbed onto or conjugated to thesurface of a microfabricated filter made of any suitable material, suchas, for example, one or more metals, one or more ceramics, one or morepolymers, glass, silica, silicon dioxide, or combinations thereof. Inpreferred embodiments of the present invention, the surface or surfacesof a microfabricated filter of the present invention is coated with acompound to increase the efficiency of filtration by reducing theinteraction of sample components with the filter surface.

For example, the surface of a filter can be coated with a molecule, suchas, but not limited to, a protein, peptide, or polymer, includingnaturally occurring or synthetic polymers. The material used to coat thefilter is preferably biocompatible, meaning it does not have deleteriouseffects on cells or other components of biological samples, such asproteins, nucleic acids, etc. Albumin proteins, such as bovine serumalbumin (BSA) are examples of proteins that can be used to coat amicrofabricated filter of the present invention. Polymers used to coat afilter can be any polymer that does not promote cell sticking to thefilter, for example, nonhydrophobic polymers such as, but not limitedto, polyethylene glycol (PEG), polyvinylacetate (PVA), andpolyvinylpyrrolidone (PVP), and a cellulose or cellulose-likederivative.

A filter made of, for example, metal, ceramics, a polymer, glass, orsilica can be coated with a compound by any feasible means, such as, forexample, adsorption or chemical conjugation.

In many cases, it can be advantageous to surface-treat the filter priorto coating with a compound or polymer. Surface treatment can increasethe stability and uniformity of the coating. For example, a filter canbe treated with at least one acid or at least one base, or with at leastone acid and at least one base, prior to coating the filter with acompound or polymer. In preferred aspects of the present invention, afilter made of a polymer, glass, or silica is treated with at least oneacid and then incubated in a solution of the coating compound for aperiod of time ranging from minutes to days. For example, a glass filtercan be incubated in acid, rinsed with water, and then incubated in asolution of BSA, PEG, or PVP.

In some aspects of the present invention, it can be preferred to rinsethe filter, such as in water (for example, deionized water) or abuffered solution before acid or base treatment or treatment with anoxidizing agent, and, preferably again before coating the filter with acompound or polymer. Where more than one type of treatment is performedon a microfabricated filter, rinses can also be performed betweentreatments, for example, between treatment with an oxidizing agent andan acid, or between treatment with an acid and a base. A filter can berinsed in water or an aqueous solution that has a pH of between about3.5 and about 10.5, and more preferably between about 5 and about 9.Nonlimiting examples of suitable aqueous solutions for rinsing iontransport measuring means can include salt solutions (where saltsolutions can range in concentration from the micromolar range to 5M ormore), biological buffer solutions, cell media, or dilutions orcombinations thereof. Rinsing can be performed for any length of time,for example from minutes to hours.

The concentration of a compound or polymer solution used to coat afilter can vary from about 0.02% to 20% or more, and will depend in parton the compound used. The incubation in coating solution can be fromminutes to days, and preferably is from about 10 minutes to two hours.

After coating, the filter can be rinsed in water or a buffer.

The treatment methods of the present invention can also be applied tochips other than those that comprise pores for filtration. For example,chips that comprises metals, ceramics, one or more polymers, silicon,silicon dioxide, or glass can be physically or chemically treated usingthe methods of the present invention. Such chips can be used, forexample, in separation, analysis, and detection devices in whichbiological species such as cells, organelles, complexes, or biomolecules(for example, nucleic acids, proteins, small molecules) are separated,detected, or analyzed. The treatment of the chip can enhance or reducethe interaction of the biological species with the chip surface,depending of the treatment used, the properties of the biologicalspecies being manipulated, and the nature of the manipulation. Forexample, a chip can be coated with a hydrophilic or hydrophobic polymer,depending on the biological species being manipulated and the nature ofthe manipulation. As a further example, coating the surface of the chipwith a hydrophilic polymer (for example but not limited to coating thechip with PVP or PVA) may reduce or minimize the interaction between thesurface of the chip and the cells.

Filter Comprising Electrodes

In some preferred embodiments, traveling-wave dielectrophoretic forcescan be generated by electrodes built onto a chip that is part of afiltration chamber, and can be used to move sample components such ascells away from a filter. In this case, the microelectrodes arefabricated onto the filter surfaces and the electrodes are arranged sothat the traveling wave dielectrophoresis can cause the samplecomponents such as cells to move on the electrode plane or the filtersurface through which the filtration process occur. A full descriptionof the traveling wave dielectrophoresis is provided in U.S. applicationSer. No. 09/679,024 having attorney docket number 471842000400, entitled“Apparatuses Containing Multiple Active Force Generating Elements andUses Thereof” filed Oct. 4, 2000, herein incorporated by reference inits entirety.

In one embodiment of the filters, interdigitated microelectrodes arefabricated onto the filter surfaces such as those shown in FIG. 2 ordescribed in “Novel dielectrophoresis-based device of the selectiveretention of viable cells in cell culture media” by Docoslis et al, inBiotechnology and Bioengineering, Vol. 54, No. 3, pages 239-250, 1997,and in the U.S. Pat. No. 5,626,734, issued to Docoslis et al. on May 7,1997. For this embodiment, the negative dielectrophoretic forcesgenerated by the electrodes can repel the sample components such as thecells from the filter surface or from the filter slots so that thecollected cells on the filters are not clogging the filters during thefiltration process. Where traveling-wave dielectrophoresis or negativedielectrophoresis is used to enhance filtration, electrode elements canbe energized periodically throughout the filtration process, duringperiods when fluid flow is halted or greatly reduced.

Filters having slots in the micron range that incorporate electrodesthat can generate dielectrophoretic forces are illustrated in FIG. 3 (Aand B). For example, filters have been made in which the interdigitatedelectrodes of 18 micron width and 18 micron gaps were fabricated on thefilters, which were made on silicon substrates. Individual filter slotswere of rectangular shape with dimensions of 100 micron (length) by2-3.8 micron (width). Each filter had a unique slot size (e.g. length bywidth: 100 micron by 2.4 micron, 100 micron by 3 micron, 100 micron by3.8 micron). Along the length direction, the gap between the adjacentfilter slots was 20 micron. Along the width direction, the adjacentslots were not aligned; instead, they were offset. The offset distancebetween neighboring columns of the filter slots were 50 micron or 30micron, alternatively. The filter slots were positioned with respect tothe electrodes so that the slot center lines along the length directionwere aligned with the center line of the electrodes, or the electrodeedges, or the center line of the gaps between the electrodes.

The following discussion and references can provide a framework for thedesign and use of electrodes to facilitate filtration by translocatingsample components, such as nonfilterable cells, away from a filter:

Dielectrophoresis refers to the movement of polarized particles in anon-uniform AC electrical field. When a particle is placed in anelectrical field, if the dielectric properties of the particle and itssurrounding medium are different, the particle will experiencedielectric polarization. Thus, electrical charges are induced at theparticle/medium interface. If the applied field is non-uniform, then theinteraction between the non-uniform field and the induced polarizationcharges will produce net force acting on the particle to cause particlemotion towards the region of strong or weak field intensity. The netforce acting on the particle is called dielectrophoretic force and theparticle motion is dielectrophoresis. Dielectrophoretic force depends onthe dielectric properties of the particles, particle surrounding medium,the frequency of the applied electrical field and the fielddistribution.

Traveling-wave dielectrophoresis is similar to dielectrophoresis inwhich the traveling-electric field interacts with the field-inducedpolarization and generates electrical forces acting on the particles.Particles are caused to move either with or against the direction of thetraveling field. Traveling-wave dielectrophoretic forces depend on thedielectric properties of the particles and their suspending medium, thefrequency and the magnitude of the traveling-field. The theory fordielectrophoresis and traveling-wave dielectrophoresis and the use ofdielectrophoresis for manipulation and processing of microparticles maybe found in various publications (e.g., “Non-uniform SpatialDistributions of Both the Magnitude and Phase of AC Electric Fieldsdetermine Dielectrophoretic Forces by Wang et al., in Biochim BiophysActa Vol. 1243, 1995, pages 185-194”, “Dielectrophoretic Manipulation ofParticles” by Wang et al, in IEEE Transaction on Industry Applications,Vol. 33, No. 3, May/June, 1997, pages 660-669, “Electrokinetic behaviorof colloidal particles in traveling electric fields: studies using yeastcells” by Huang et al, in J. Phys. D: Appl. Phys., Vol. 26, pages1528-1535, “Positioning and manipulation of cells and microparticlesusing miniaturized electric field traps and traveling waves” By Fuhr etal., in Sensors and Materials. Vol. 7: pages 131-146, “Dielectrophoreticmanipulation of cells using spiral electrodes” by Wang, X-B. et al., inBiophys. J. Volume 72, pages 1887-1899, 1997, “Separation of humanbreast cancer cells from blood by differential dielectric affinity” byBecker et al, in Proc. Natl. Acad. Sci., Vol., 92, January 1995, pages860-864). The manipulation of microparticles with dielectrophoresis andtraveling wave dielectrophoresis include concentration/aggregation,trapping, repulsion, linear or other directed motion, levitation,separation of particles. Particles may be focused, enriched and trappedin specific regions of the electrode reaction chamber. Particles may beseparated into different subpopulations over a microscopic scale.Relevant to the filtration methods of the present invention, particlesmay be transported over certain distances. The electrical fielddistribution necessary for specific particle manipulation depends on thedimension and geometry of microelectrode structures and may be designedusing dielectrophoresis theory and electrical field simulation methods.

The dielectrophoretic force F_(DEP) _(z) acting on a particle of radiusr subjected to a non-uniform electrical field can be given by

F _(DEP) _(z) =2π∈_(m) r ³χ_(DEP) ∇E _(rms) ² ·{right arrow over (a)}_(z)

where E_(rms) is the RMS value of the field strength, ∈_(m) is thedielectric permittivity of the medium. χ_(DEP) is the particledielectric polarization factor or dielectrophoresis polarization factor,given by

${\chi_{DEP} = {{Re}( \frac{ɛ_{p}^{*} - ɛ_{m}^{*}}{ɛ_{p}^{*} + {2ɛ_{m}^{*}}} )}},$

“Re” refers to the real part of the “complex number”. The symbol

$ɛ_{x}^{*} = {ɛ_{x} - {j\frac{\sigma_{x}}{2\pi \; f}}}$

is the complex permittivity (of the particle x=p, and the medium x=m).The parameters ∈_(p) and σ_(p) are the effective permittivity andconductivity of the particle, respectively. These parameters may befrequency dependent. For example, a typical biological cell will havefrequency dependent, effective conductivity and permittivity, at least,because of cytoplasm membrane polarization.

The above equation for the dielectrophoretic force can also be writtenas

F _(DEP) _(z) =2π∈_(m) r ³χ_(DEP) V ² p(z){right arrow over (a)} _(z)

where p(z) is the square-field distribution for a unit-voltageexcitation (V=1 V) on the electrodes, V is the applied voltage.

There are generally two types of dielectrophoresis, positivedielectrophoresis and negative dielectrophoresis. In positivedielectrophoresis, particles are moved by dielectrophoresis forcestowards the strong field regions. In negative dielectrophoresis,particles are moved by dielectrophoresis forces towards weak fieldregions. Whether particles exhibit positive and negativedielectrophoresis depends on whether particles are more or lesspolarizable than the surrounding medium. In the filtration methods ofthe present invention, electrode patterns on one or more filters of afiltration chamber can be designed to cause sample components such ascells to exhibit negative dielectrophoresis, resulting in samplecomponents such as cells being repelled away from the electrodes on thefilter surfaces.

Traveling-wave DEP force refers to the force that is generated onparticles or molecules due to a traveling-wave electric field. Atraveling-wave electric field is characterized by the non-uniformdistribution of the phase values of AC electric field components.

Here we analyze the traveling-wave DEP force for an ideal traveling-wavefield. The dielectrophoretic force F_(DEP) acting on a particle ofradius r subjected to a traveling-wave electrical field E_(TWD)=Ecos(2π(ft−z/λ₀)){right arrow over (a)}_(x) (i.e., a x-direction field istraveling along the z-direction) is given by

F _(TWD)=−2π∈_(m) r ³ζ_(TWD) E ² ·{right arrow over (a)} _(z)

where E is the magnitude of the field strength, ∈_(m) is the dielectricpermittivity of the medium. ζ_(TWD) is the particle polarization factor,given by

${\zeta_{TWD} = {{Im}( \frac{ɛ_{p}^{*} - ɛ_{m}^{*}}{ɛ_{p}^{*} + {2ɛ_{m}^{*}}} )}},$

“Im” refers to the imaginary part of the “complex number”. The symbol

$ɛ_{x}^{*} = {ɛ_{x} - {j\frac{\sigma_{x}}{2\pi \; f}}}$

is the complex permittivity (of the particle x=p, and the medium x=m).The parameters ∈_(p) and σ_(p) are the effective permittivity andconductivity of the particle, respectively. These parameters may befrequency dependent.

Particles such as biological cells having different dielectric property(as defined by permittivity and conductivity) will experience differentdielectrophoretic forces. For traveling-wave DEP manipulation ofparticles (including biological cells), traveling-wave DEP forces actingon a particle of 10 micron in diameter can vary somewhere between 0.01and 10000 pN.

A traveling wave electric field can be established by applyingappropriate AC signals to the microelectrodes appropriately arranged ona chip. For generating a traveling-wave-electric field, it is necessaryto apply at least three types of electrical signals each having adifferent phase value. An example to produce a traveling wave electricfield is to use four phase-quardrature signals (0, 90, 180 and 270degrees) to energize four linear, parallel electrodes patterned on thechip surfaces. Such four electrodes form a basic, repeating unit.Depending on the applications, there may be more than two such unitsthat are located next to each other. This will produce atraveling-electric field in the spaces above or near the electrodes. Aslong as electrode elements are arranged following certain spatiallysequential orders, applying phase-sequenced signals will result inestablishing traveling electrical fields in the region close to theelectrodes.

Both dielectrophoresis and traveling-wave dielectrophoresis forcesacting on particles depend on not only the field distributions (e.g.,the magnitude, frequency and phase distribution of electrical fieldcomponents; the modulation of the field for magnitude and/or frequency)but also the dielectric properties of the particles and the medium inwhich particles are suspended or placed. For dielectrophoresis, ifparticles are more polarizable than the medium (e.g., having largerconductivities and/or permittivities depending on the appliedfrequency), particles will experience positive dielectrophoresis forcesand are directed towards the strong field regions. The particles thatare less polarizable than the surrounding medium will experiencenegative dielectrophoresis forces and are directed towards the weakfield regions. For traveling wave dielectrophoresis, particles mayexperience dielectrophoresis forces that drive them in the samedirection as the field traveling direction or against it, dependent onthe polarization factor ζ_(TWD). The following papers provide basictheories and practices for dielectrophoresis andtraveling-wave-dielectrophoresis: Huang, et al., J. Phys. D: Appl. Phys.26:1528-1535 (1993); Wang, et al., Biochim. Biophys. Acta. 1243:185-194(1995); Wang, et al., IEEE Trans. Ind. Appl. 33:660-669 (1997).

Filtration Chamber

A filtration chamber or the present invention is any chamber that cancontain a fluid sample that comprises or engages at least onemicrofabricated filter of the present invention. A filtration chamber ofthe present invention can comprise one or more fluid-impermeablematerials, such as but not limited to, metals, polymers, plastics,ceramics, glass, silicon, or silicon dioxide. Preferably, a filtrationchamber of the present invention has a volumetric capacity of from about0.01 milliliters to about ten liters, more preferably from about 0.2milliliters to about two liters. In some preferred embodiments of thepresent invention, a filtration chamber can have a volume of from about1 milliliter to about 80 milliliters.

A filtration chamber of the present invention can comprise or engage anynumber of filters. In one preferred embodiment of the present invention,a filtration chamber comprises one filter (see, for example FIG. 5 andFIG. 14. In another preferred embodiment of the present invention, afiltration chamber comprises more than one filter, such as the chamberexemplified in FIG. 6 and FIG. 7. Various filter chamber configurationsare possible. For example, it is within the scope of the presentinvention to have a filtration chamber in which one or more walls of thefilter chamber comprises a microfabricated filter. It is also within thescope of the present invention to have a filtration chamber in which afilter chamber engages one or more filters. In this case, the filterscan be permanently engaged with the chamber, or can be removable (forexample, they can be inserted into slots or tracks provided on thechamber). A filter can be provided as a wall of a chamber, or internalto a chamber, and filters can optionally be provided in tandem forsequential filtering. Where filters are inserted into a chamber, theyare inserted to form a tight seal with the walls of a chamber, such thatduring the filtration operation, fluid flow through the chamber (fromone side of a filter to the other) must be through the pores of thefilter.

In embodiments in which a filtration chamber of the present inventioncomprises one or more microfabricated filters that are internal to thechamber, the filter or filters can divide the chamber into subchambers.Where a filtration chamber comprises a single internal microfabricatedfilter, for example, the filtration chamber can comprise a prefiltration“antechamber”, or where appropriate, “upper subchamber” and a“post-filtration subchamber”, or, where appropriate, “lower subchamber”.In other cases, a microfabricated filter can form a wall of a filtrationchamber, and during filtration, filterable sample components exit thechamber via the filter.

In some preferred embodiments of the present invention, a filtrationchamber of the present invention has at least one port that allows forthe introduction of a sample into the chamber, and conduits cantransport sample to and from a filtration chamber of the presentinvention. When fluid flow commences, sample components that flowthrough one or more filters can flow into one or more areas of thechamber and then out of the chamber through conduits, and, preferablybut optionally, from the conduits into a vessel, such as a waste vessel.The filtration chamber can also optionally have one or more additionalports for the additions of one or more reagents, solutions, or buffers.

In some preferred embodiments, a filtration chamber of the presentinvention is part of a filtration unit in which valves control fluidflow through the chamber. For example, one preferred filtration unit ofthe present invention, depicted in FIG. 5, comprises a valve-controlledinlet for the addition of sample (valve A (6)), a valve connected to aconduit through which negative pressure is applied for the filtration ofthe sample (valve B (7)), and a valve controlling the flow of washbuffer into the filtration chamber for washing the chamber (valve C(8)). In some preferred embodiments of the present invention, afiltration unit can comprise valves that can optionally be underautomatic control that allow sample to enter the chamber, waste to exitthe chamber, and negative pressure to provide fluid flow for filtration.

In order to transfer a solution or supernatant to the filtrationchamber, a needle (but not limited to stated object) can be used. Aneedle may be connected to the container (e.g. tubing or chamber) thatcan hold a volume. The needle may collect cells from a tube containing asolution and dispenses the solution into another chamber using a deviceto push or pull a solution (e.g. pump or syringe).

The chamber may include one or more surface contours to affect the flowof a sample, a solution such as wash or elution solution or both. Forexample contours may deflect, disperse or direct a sample to assist inthe spreading of the sample along the chip. Alternatively, contours maydeflect, disperse or direct a wash solution such that the wash solutionwashes the chamber or chip with greater efficiency. Such surfacecontours may be in any appropriate configuration. The contours mayinclude surfaces that project generally toward the chip or may projectgenerally away from the chip. They may generally encircle the chip.Contours may include but are not limited to projections, recessedportions, slots, deflection structures such as ball-like portions,bubbles (formed from e.g. air, detergent, or polymers), and the like.Contours such as two or more slots may be configured generally parallelto one another yet generally angled when viewing the chamber upright todirect flow in a generally spiraled path.

In a preferred embodiment of the present invention, a filtration chamberof, for example, approximately one centimeter by one centimeter by 0.2to ten centimeters in dimensions can have one or more filters comprisingfrom four to 1,000,000 slots, preferably from 100 to 250,000 slots. Inthis preferred embodiment, the slots are preferably of rectangularshape, with a slot length of from about 0.1 to about 1,000 microns, andslot width is preferably from about 0.1 to about 100 microns, dependingon the application.

Preferably, slots can allow for the passage of mature red blood cells(lacking nuclei) through the channels and thus out of the chamber, whilenot or minimally allowing cells having a greater diameter or shape (forexample but not limited to, nucleated cells such as white blood cellsand nucleated red blood cells) to exit the chamber. A filtration chamberthat can allow the removal of red blood cells by fluid flow through thechamber, while retaining other cells of a blood sample, is illustratedin FIG. 7, FIG. 14, and FIG. 16. For example, for removing matured redblood cells from nucleated RBCs and white blood cells, slot widthsbetween 2.5 and 6.0 microns, more preferably between 2.5 and 4.0microns, could be used. Slot length could vary between, for example, 20and 200 microns. Slot depth (i.e., filter membrane thickness) can varybetween 40 and 100 microns. The slot width between 2.0 and 4.0 micronswould allow the double-discoid-shaped RBCs to go through the slots whileprimarily retaining the nucleated RBCs and WBCs with diameters or shapeslarger than 7 micron.

Filtration Chamber Comprising Active Chip

A filtration chamber can also preferably comprise or engage at least aportion of at least one active chip, where an active chip is a chip thatuses applied physical forces to promote, enhance, or facilitateprocessing or desired biochemical reactions of a sample, or and todecrease or reduce any undesired effects that might otherwise occur toor in a sample. An active chip of a filtration chamber of the presentinvention preferably comprises acoustic elements, electrodes, or evenelectromagnetic elements. An active chip can be used to transmit aphysical force that can prevent clogging of the slots or around thestructures used to create a filter (for example, blocks, dams, orchannels, slots etched into and through the filter substrate) bycomponents of the sample that are too large to go through the pores orslots or openings, or become aggregated at the pores or slots oropenings. For example, when an electric signal is applied, acousticelements can cause mixing of the components within the chamber, therebydislodging nonfilterable components from the slots or pores. In analternative embodiment, a pattern of electrodes on a chip can providenegative dielectrophoresis of sample components to move thenonfilterable components from the vicinity of the slots, channels, oropenings around structures and allow access of filterable samplecomponents to the slots or openings. Example of such electrode arraysfabricated onto a filter under a different operating mechanism of“dielectrophoretic-base selective retention” have been described in“Novel dielectrophoresis-based device of the selective retention ofviable cells in cell culture media” by Docoslis et al, in Biotechnologyand Bioengineering, Vol. 54, No. 3, pages 239-250, 1997, hereinincorporated by reference and in the U.S. Pat. No. 5,626,734, issued toDocoslis et al on May 7, 1997, herein incorporated by reference. Activechips, including chips that can be used to mix samples by acousticforces and chips that can be used to move moieties, including samplecomponents, by dielectrophoretic forces, are described in U.S.application Ser. No. 09/636,104, filed Aug. 10, 2000, entitled “Methodsfor Manipulating Moieties in Microfluidic Systems”, U.S. provisionalapplication 60/239,299, entitled “An Integrated Biochip System forSample Preparation and Analysis”, filed Oct. 10, 2000, and U.S.application Ser. No. 09/686,737, filed Oct. 10, 2000 entitled“Compositions and Methods for Separation of Moieties on Chips”, allherein incorporated by reference.

The incorporation of electrodes that can be used for traveling wavedielectrophoresis on a filter of the present invention, as well asprinciples of dielectrophoresis and traveling wave dielectrophoresis,has been described herein in a previous description of microfabricatedfilters. Electrodes can also be incorporated onto active chips that areused in filtration chambers of the present invention to improvefiltration efficiency.

A filtration chamber can also comprise a chip that compriseselectromagnetic elements. Such electromagnetic elements can be used forthe capture of sample components before or, preferably, after, filteringof the sample. Sample components can be captured after being bound tomagnetic beads. The captured sample components can be either undesirablecomponents to be retained in the chamber after the sample containingdesirable components has already been removed from the chamber, or thecaptured sample components can be desirable components captured in thechamber after filtration.

An acoustic force chip can engage or be part of a filtration chamber, orone or more acoustic elements can be provided on one or more walls of afiltration chamber. Mixing of a sample by the activation of the acousticforce chip can occur during the filtration procedure. Preferably, apower supply is used to transmit an electric signal to the acousticelements of one or more acoustic chips or one or more acoustic elementson one or more walls or a chamber. One or more acoustic elements can beactive continuously throughout the filtration procedure, or can beactivated for intervals (pulses) during the filtration procedure.

Sample components and, optionally, solutions or reagents added to thesample can be mixed by acoustic forces that act on both the fluid andthe moieties, including, but not limited to, molecules, complexes,cells, and microparticles, in the chamber. Acoustic forces can causemixing by acoustic streaming of fluid that occurs when acousticelements, when energized by electrical signals generate mechanicalvibrations that are transmitted into and through the fluid. In addition,acoustic energy can cause movement of sample components and/or reagentsby generating acoustic waves that generate acoustic radiation forces onthe sample components (moieties) or reagents themselves.

The following discussion and references can provide a framework for thedesign and use of acoustic elements to provide a mixing function:

Acoustic force refers to the force that is generated on moieties, e.g.,particles and/or molecules, by an acoustic wave field. (It may also betermed acoustic radiation forces.) The acoustic forces can be used formanipulating, e.g., trapping, moving, directing, handling, mixing,particles in fluid. The use of the acoustic force in a standingultrasound wave for particle manipulation has been demonstrated forconcentrating erythrocytes (Yasuda et al, J. Acoust. Soc. Am.,102(1):642-645 (1997)), focusing micron-size polystyrene beads (0.3 to10 micron in diameter, Yasuda and Kamakura, Appl. Phys. Lett,71(13):1771-1773 (1997)), concentrating DNA molecules (Yasuda et al, J.Acoust. Soc. Am., 99(2):1248-1251, (1996)), batch and semicontinuousaggregation and sedimentation of cells (Pui et al, Biotechnol. Prog.,11:146-152 (1995)). By competing electrostatic and acoustic radiationforces, separation of polystyrene beads of different size and chargeshave been reported (Yasuda et al, J. Acoust. Soc. Am., 99(4):1965-1970(1996); and Yasuda et al., Jpn. J. Appl. Phys., 35(1):3295-3299 (1996)).Furthermore, little or no damage or harming effect was observed whenacoustic radiation force was used to manipulate mammalian cells, ascharacterized in terms of ion leakage (for erythrocytes, Yasuda et al,J. Acoust. Soc. Am., 102(1):642-645 (1997)) or antibody production (forhybridoma cells, Pui et al, Biotechnol. Prog., 11:146-152 (1995)).

An acoustic wave can be established by an acoustic transducer, e.g.,piezoelectric ceramics such as PZT material. The piezoelectrictransducers are made from “piezoelectric materials” that produce anelectric field when exposed to a change in dimension caused by animposed mechanical force (piezoelectric or generator effect).Conversely, an applied electric field will produce a mechanical stress(electrostrictive or motor effect) in the materials. They transformenergy from mechanical to electrical and vice-versa. When AC voltagesare applied to the piezoelectric transducers, the vibration occurs tothe transducers and such vibration can be coupled into a fluid that isplaced in the chamber comprising the piezoelectric transducers.

An acoustic chip can comprise acoustic transducers so that when ACsignals at appropriate frequencies are applied to the electrodes on theacoustic transducers, the alternating mechanical stress is producedwithin the piezoelectric materials and is transmitted into the liquidsolutions in the chamber. In a situation where the chamber is set up sothat a standing acoustic wave is established along the direction (e.g.:z-axis) of wave propagation and reflection, the standing wave spatiallyvarying along the z axis in a fluid can be expressed as:

Δp(z)=p ₀ sin(kz)cos(ωt)

where Δp is acoustic pressure at z, p₀ is the acoustic pressureamplitude, k is the wave number (2π/λ, where λ is the wavelength), z isthe distance from the pressure node, ω is the angular frequency, and tis the time. In one example, the standing-wave acoustic field may begenerated by the superimposition of an acoustic wave generated from anacoustic transducer that forms a major surface of a chamber and thereflective wave from another major surface of the chamber that ispositioned in parallel with the acoustic transducer and reflects theacoustic wave from the transducer. According to the theory developed byYosioka and Kawasima (Acoustic Radiation Pressure on a CompressibleSphere by Yosioka K. and Kawasima Y. in Acustica, Volume 5, pages167-173, 1955), the acoustic force F_(acoustic) acting on a sphericalparticle in the stationary standing wave field is given by

$F_{acoustic} = {{- \frac{4\pi}{3}}r^{3}{kE}_{acoustic}A\; {\sin ( {2{kz}} )}}$

where r is the particle radius, E_(acoustic) is the average acousticenergy density, A is a constant given by

$A = {\frac{{5\rho_{p}} - {2\rho_{m}}}{{2\rho_{p}} + \rho_{m}} - \frac{\gamma_{p}}{\gamma_{m}}}$

where ρ_(m) and ρ_(p) are the density of the particle and the medium,γ_(m) and γ_(p) are the compressibility of the particle and medium,respectively. The compressibility of a material is the product of thedensity of the material and the velocity of acoustic-wave in thematerial. The compressibility is sometimes termed acoustic impedance. Ais termed as the acoustic-polarization-factor.

When A>0, the particle moves towards the pressure node (z=0) of thestanding wave.

When A<0, the particle moves away from the pressure node.

The acoustic radiation forces acting on particles depend on acousticenergy density distribution and on particle density and compressibility.Particles having different density and compressibility will experiencedifferent acoustic-radiation-forces when they are placed into the samestanding acoustic wave field. For example, the acoustic radiation forceacting on a particle of 10 micron in diameter can vary somewhere between<0.01 and >1000 pN, depending on the established acoustic energy densitydistribution.

The above analysis considers the acoustic radiation forces exerted onparticles in a standing acoustic wave. Further analysis may be extendedto the case of the acoustic radiation forces exerted on particles in atraveling-wave case. Generally, an acoustic wave field may consist ofboth standing and traveling-wave components. In such cases, particles inthe chamber will experience acoustic radiation forces in the form otherthan those described by above equations. The following papers providedetailed analysis of acoustic radiation forces on spherical particles bytraveling acoustic wave and standing acoustic waves: “Acoustic RadiationPressure on a Compressible Sphere” by Yosioka K. and Kawasima Y. inAcustica, Volume 5, pages 167-173, 1955; and “Acoustic-Radiation forceon a solid elastic sphere” by Hasegawa T. and Yosioka K. in Journal ofAcoustic Society of America.

The acoustic radiation forces on particles may also be generated byvarious special cases of acoustic waves. For example, acoustic forcesmay be produced by a focused beam (“Acoustic radiation force on a smallcompressible sphere in a focused beam” by Wu and Du, J. Acoust. Soc.Am., 87:997-1003 (1990)), or by acoustic tweezers (“Acoustic tweezers”by Wu J. Acoust. Soc. Am., 89:2140-2143 (1991)).

Acoustic wave field established in a fluid can also induce atime-independent fluid flow, as termed acoustic streaming. Such fluidflow may also be utilized in biochip applications or microfluidicapplications for transporting or pumping fluids. Furthermore, suchacoustic-wave fluid flow may be exploited for manipulating molecules orparticles in fluids. The acoustic streaming depends on acoustic fielddistributions and on fluid properties (“Nonlinear phenomena” by RooneyJ. A. in “Methods of Experimental Physics: Ultrasonics, Editor: P. D.Edmonds”, Chapter 6.4, pages 319-327, Academic Press, 1981; “AcousticStreaming” by Nyborg W. L. M. in “Physical Acoustics”, Vol. II-Part B,Properties of Polymers and Nonlinear Acoustics, Chapter 11, pages265-330).

Thus, one or more active chips, such as one or more acoustic forcechips, can also be used to promote mixing of reagents, solutions, orbuffers, that can be added to a filtration chamber, before, during, orafter the addition of a sample and the filtration process. For example,reagents, such as, but not limited to specific binding members that canaid in the removal of undesirable sample components, or in the captureof desirable sample components, can be added to a filtration chamberafter the filtration process has been completed and the conduits havebeen closed off. The acoustic elements of the active chip can be used topromote mixing of one or more specific binding members with the samplewhose volume has been reduced by filtration. One example is the mixingof sample components with magnetic beads that comprise antibodies thatcan bind particular cell types (for example, white blood cells, or fetalnucleated red blood cells) within the sample. The magnetic beads can beused to selectively remove or separate (capture) undesirable ordesirable sample components, respectively, in subsequent steps of amethod of the present invention. The acoustic elements can be activatedfor a continuous mixing period, or in pulses.

II. Method of Enriching Rare Cells of a Fluid Sample UsingMicrofiltration

The present invention provides methods of enriching rare cells of afluid sample using filtration through a microfabricated filter of thepresent invention that comprises at least one tapered pore. The methodincludes: dispensing a sample into a filtration chamber that comprisesor engages at least one microfabricated filter that comprises at leastone tapered pore; providing fluid flow of the sample through thefiltration chamber, such that components of the fluid sample flowthrough or are retained by the one or more microfabricated filters basedon the size, shape, or deformability of the components; and collectingenriched rare cells from said filtration chamber. In some embodiments,filtration can separate soluble and small components of a sample from atleast a portion of the cells that are in the sample, in order toconcentrate the retained cells to facilitate further separation andanalysis. In some aspects, filtration can remove undesirable componentsfrom a sample, such as, but not limited to, undesirable cell types.Where filtration reduces the volume of a sample by at least 50% orremoves greater than 50% of the cellular components of a sample,filtration can be considered a debulking step. The present inventioncontemplates the use of filtration for debulking as well as otherfunctions in the processing of a fluid sample, such as, for example,concentration of sample components or separation of sample components(including, for example, removal of undesirable sample components andretention of desirable sample components).

Sample

A sample can be any fluid sample, such as an environmental sample,including air samples, water samples, food samples, and biologicalsamples, including suspensions, extracts, or leachates of environmentalor biological samples. Biological samples can be blood, a bone marrowsample, an effusion of any type, ascities fluid, pelvic wash fluid, orpleural fluid, spinal fluid, lymph, serum, mucus, sputum, saliva, urine,semen, occular fluid, extracts of nasal, throat or genital swabs, cellsuspension from digested tissue, or extracts of fecal material.Biological samples can also be samples of organs or tissues, includingtumors, such as fine needle aspirates or samples from perfusions oforgans or tissues. Biological samples can also be samples of cellcultures, including both primary cultures and cell lines. The volume ofa sample can be very small, such as in the microliter range, and mayeven require dilution, or a sample can be very large, such as up toabout two liters for ascites fluid. A preferred sample is a bloodsample.

A blood sample can be any blood sample, recently taken from a subject,taken from storage, or removed from a source external to a subject, suchas clothing, upholstery, tools, etc. A blood sample can therefore be anextract obtained, for example, by soaking an article containing blood ina buffer or solution. A blood sample can be unprocessed or partiallyprocessed, for example, a blood sample that has been dialyzed, hadreagents added to it, etc. A blood sample can be of any volume. Forexample, a blood sample can be less than five microliters, or more than5 liters, depending on the application. Preferably, however, a bloodsample that is processed using the methods of the present invention willbe from about 10 microliters to about 2 liters in volume, morepreferably from about one milliliter to about 250 milliliters in volume,and most preferably between about 5 and 50 milliliters in volume.

The rare cells to be enriched from a sample can be of any cell typepresent at less than one million cells per milliliter of fluid sample orthat constitute less than 1% of the total nucleated cell population in afluid sample. Rare cells can be, for example, bacterial cells, fungalcells, parasite cells, cells infected by parasites, bacteria, orviruses, or eukaryotic cells such as but not limited to fibroblasts orblood cells. Rare blood cells can be RBCs (for example, if the sample isan extract or leachate containing less than one million red blood cellsper milliliter), subpopulations of blood cells and blood cell types,such as WBCs, or subtypes of WBCs (for example, T cells or macrophages),nucleated red blood cells, or can be fetal cells (including but notlimited to nucleated red blood cells, trophoblasts, granulocytes, ormonocytes). Rare cells can be stem or progenitor cells of any type. Rarecells can also be cancer cells, including but not limited to neoplasticcells, malignant cells, and metastatic cells. Rare cells may becandidate cancer cells, such as a rare cell in a blood sample that couldbe enriched and subjected to the process of detection, identification,characterization and the like that may specify a cancer cell. Rare cellsof a blood sample can also be non-hematopoietic cells, such as but notlimited to epithelial cells.

Dispensing of Sample into Filtration Chamber

A sample can be dispensed into a filtration chamber of the presentinvention by any convenient means. As nonlimiting examples, sample canbe introduced using a conduit (such as tubing) through which a sample ispumped or injected into the chamber, or can be directly poured,injected, or dispensed or pipeted manually, by gravity feed, or by amachine. Dispensing of a sample into a filtration chamber of the presentinvention can be directly into the filtration chamber, via a loadingreservoir that feeds directly or indirectly into a filtration chamber,or can be into a conduit that leads to a filtration chamber, or into avessel that leads, via one or more conduits, to a filtration chamber. Aneedle (or any fluid drawing device) in fluid communication with tubingor a chamber can also be used to enter a tube. The needle may collectcells from a tube containing a solution and dispense the solution intoanother chamber using a device to push or pull a solution (e.g. pump orsyringe).

Filtering

Following the addition to a filtration chamber of the present invention,filtering is effected by providing fluid flow through the chamber. Fluidflow can be provided by any means, including positive or negativepressure (for example, by a manual or machine operated syringe-typesystem), pumping, or even gravity. The filtration chamber can have portsthat are connected to conduits through which a buffer or solution andthe fluid sample or components thereof can flow. A filtration unit canalso have valves that can control fluid flow through the chamber. Whenthe sample is added to the filtration chamber, and fluid flow isdirected through the chamber, filter slots can allow the passage offluid, soluble components of the samples, and filterable non-solublecomponents of a fluid sample through a filter, but, because of the slotdimensions, can prevent the passage of other components of the fluidsample through the filter.

Preferably, fluid flow through a filtration chamber of the presentinvention is automated, and performed by a pump or positive or negativepressure system, but this is not a requirement of the present invention.The optimal flow rate will depend on the sample being filtered,including the concentration of filterable and nonfilterable componentsin the sample and their ability to aggregate and clog the filter. Forexample, the flow rate through the filtration chamber can be from lessthan 1 milliliter per hour to more than 1000 milliliters per hour, andflow rate is in no way limiting for the practice of the presentinvention. Preferably, however, filtration of a blood sample occurs at arate of from 5 to 500 milliliters per hour, and more preferably at arate of between about 10 and about 50 milliliters per hour.

In fabricating the filter slots through the filter substrate, slighttapering of the slot along the slot depth direction can occur. Thus aparticular slot width may not be maintained constant throughout theentire depth of the filter and the slot width on one surface of thefilter is typically larger than the width on the opposite surface. Inutilizing such filters with tapered slot width, it is preferred to havethe narrow-slot side of the filter facing the sample, so that duringfiltering the sample goes through the narrow-width side of the slotfirst and then filtered cells exit at the wide-width side of the slot.This avoids trapping cells that are being filtered within thefunnel-shaped slots. However, the orientation of a filter with one ormore tapered slots is not a restriction in using the filters of thepresent invention. Depending on specific applications, the filters canalso be used in the orientation such that the wide-width side of thefilter slots faces the sample.

In the methods of the present invention, preferably desirablecomponents, such as rare cells whose enrichment is desired, are retainedby the filter. Preferably, in the methods of the present invention asrare cells of interest of the sample are retained by the filter and oneor more undesirable components of the sample flow through the filter,thereby enriching the rare cells of interest of the sample by increasingthe proportion of the rare cells to total cells in the filter-retainedportion of the sample, although that is not a requirement of the presentinvention. For example, in some embodiments of the present invention,filtration can enrich rare cells of a fluid sample by reducing thevolume of the sample and thereby concentrating rare cells.

After filtering of the sample, optionally buffer can be washed throughthe filtration chamber to wash through any residual filterable cells.The buffer can be conveniently directed through the filtration chamberin the same manner as the sample, that is, preferably by automated fluidflow such as by a pump or pressure system, or by gravity, or the buffercan use a different fluid flow means that the sample. Typically thespeed at which the wash buffer flows through the chamber will be greaterthan that of a sample, but this need not be the case. One or more washescan be performed, using the same or different wash buffers. In addition,optionally air can be forced through the filtration chamber, for exampleby positive pressure or pumping, to push residual cells through thefiltration chamber. Also, it is possible to have one or more washes backflushed into the filtration chamber to assist in the washing of thechamber or removal of undesirable cells or assist in the recovery ofdesirable cells.

Additional Enrichment Steps

The present invention also contemplates using filtration in combinationwith other steps that can be used in enriching rare cells of a fluidsample. For example, debulking steps or separation steps can be usedprior to or following filtration, such as but not limited to asdisclosed in U.S. patent application Ser. No. 10/701,684, entitled“Methods, Compositions, and Automated Systems for Separating Rare Cellsfrom Fluid Samples” filed Nov. 4, 2003, U.S. patent application Ser. No.10/268,312, entitled “Methods, Compositions, and Automated Systems forSeparating Rare Cells from Fluid Samples” filed Oct. 10, 2002, both ofwhich are incorporated herein by reference for all disclosure relatingto debulking and separation procedures that can be used in enrichingrare cells of a fluid sample.

III. Methods of Enriching Rare Cells from a Blood Sample

The present invention includes novel and improved designs and methodsfor isolating rare cells from a blood sample. Blood sample preparationand rare cell enrichment methods known in the art and disclosed U.S.patent application Ser. No. 10/701,684, filed Nov. 4, 2003, U.S. patentapplication Ser. No. 10/268,312, filed Oct. 10, 2002, herebyincorporated by reference for all disclosure of blood sample preparationand rare cell isolation from blood samples, can be combined with themethods and designs disclosed herein.

Maternal Blood Sample Selection for Fetal Cell Isolation

The present invention includes methods for rare cell isolation fromblood samples that include the selection of a blood sample of aparticular gestational age for isolation of particular fetal cell types.

In one preferred embodiment of the present invention, a maternal bloodsample for the isolation of fetal nucleated cells is selected to be fromthe gestational age of between about 4 weeks and about 37 weeks,preferably about 7 weeks and about 24 weeks, and more preferably betweenabout 10 weeks and about 20 weeks. In this embodiment, a maternal bloodsample for the isolation of fetal nucleated cells is drawn from apregnant subject at the gestational age of between about 4 weeks andabout 37 weeks, preferably about 7 weeks and about 24 weeks, and morepreferably between about 10 weeks and about 20 weeks. As used herein, apregnant subject can also include a woman of the given gestational agethat has aborted within twenty-four hours of the blood sample draw.

Use of the Second Wash Supernatant for Isolation Fetal Cells from aMaternal Blood Sample

The present invention also includes methods for isolating fetal cellsfrom a maternal blood sample in which the supernatant of a secondcentrifugation performed on the blood sample to wash the cells prior toa debulking or separation step is used as at least a part of the samplefrom which fetal cells are isolated.

Use of an Antibody to Remove Platelets from a Blood Sample

The present invention also includes the use of an antibody or moleculecapable of specifically binding a platelet or a molecule associated witha platelet. As a nonlimiting example, antibodies or molecules or thepresent invention may specifically bind CD31, CD36, CD41, CD42(a,b,c),CD51 or CD51/61. CD31 is an endothelial and platelet cell marker thathas minimal binding to fetal cells. Its use in separating platelets froma blood sample is described in the examples.

Improved Magnet Configurations for Capture of Sample Components

A debulked sample, such as a debulked blood sample, can be incubatedwith one or more specific binding members, such as, but not limited to,antibodies, that specifically recognize one or more undesirablecomponents of a fluid sample. Where a filtration chamber has been usedfor debulking the sample, mixing and incubation of one or more specificbinding members with the sample can optionally be performed in afiltration chamber. The one or more undesirable components can becaptured, either directly or indirectly, via their binding to thespecific binding member. For example, a specific binding member can bebound to a solid support, such as a bead, membrane, or column matrix,and following incubation of the fluid sample with the specific bindingmember, the fluid sample, containing unbound components, can be removedfrom the solid support. Alternatively, one or more primary specificbinding members can be incubated with the fluid sample, and, preferablyfollowing washing to remove unbound specific binding members, the fluidsample can be contacted with a secondary specific binding member thatcan bind or is bound to a solid support. In this way the one or moreundesirable components of the sample can become bound to a solidsupport, enabling separation of the undesirable components from thefluid sample.

In a preferred aspect of the present invention, a debulked blood samplefrom a pregnant individual is incubated with magnetic beads that arecoated with antibody that specifically binds white blood cells and doesnot appreciably bind fetal nucleated cells. The magnetic beads arecollected using capture by activated electromagnetic units (such as onan electromagnetic chip), or capture by at least one permanent magnetthat is in physical proximity to a vessel, such as a tube or column,that contains the fluid sample. After capture of the magnetic beads bythe magnet, the remaining fluid sample is removed from the vessel. Thesample can be removed manually, such as by pipetting, or by physicalforces such as gravity, or by fluid flow through a separation column. Inthis way, undesirable white blood cells can be selectively removed froma maternal blood sample. The sample can optionally be further filteredusing a microfabricated filter of the present invention. Filtrationpreferably removes residual red blood cells from the sample and can alsofurther concentrate the sample.

In one preferred embodiment, after incubation of magnetic beads thatcomprise a specific binding member that specifically bind undesirablecomponents with a sample, the sample is transported through a separationcolumn that comprises or engages at least one magnet. As the sampleflows through the column, undesirable components that are bound to themagnetic beads adhere to one or more walls of the tube adjacent to themagnet or magnets. An alternative embodiment uses a magnetic separator,such as the magnetic separator manufactured by Immunicon. Magneticcapture can also employ electromagnetic chips that compriseelectromagnetic physical force-generating elements, such as thosedescribed in U.S. Pat. No. 6,355,491 entitled “Individually AddressableMicro-Electromagnetic Unit Array Chips” issued Mar. 12, 2002 to Zhou etal., U.S. application Ser. No. 09/955,343 having attorney docket numberART-00104.P.2, filed Sep. 18, 2001, entitled “Individually AddressableMicro-Electromagnetic Unit Array Chips” and U.S. application Ser. No.09/685,410 having attorney docket number ART-00104.P.1.1, filed Oct. 10,2000, entitled “Individually Addressable Micro-Electromagnetic UnitArray Chips in Horizontal Configurations”. In yet another preferredembodiment, a tube that contains the sample and magnetic beads ispositioned next to one or more magnets for the capture of nondesirablecomponents bound to magnetic beads. The supernatant, depleted of the oneor more nondesirable components, can be removed from the tube after thebeads have collected at the tube wall.

In some preferred embodiments of the present invention, removal of whiteblood cells from a sample is performed simultaneously with debulking theblood sample by selective sedimentation of red blood cells. In theseembodiments, a solution that selectively sediments red blood cells isadded to a blood sample, and a specific binding member that specificallybinds white blood cells that is bound to a solid support, such asmagnetic beads, is added to the blood sample. After mixing, red bloodcells are allowed to settle, and white blood cells are captured, such asby magnetic capture. This can be conveniently performed in a tube towhich a sedimenting solution and the specific binding member, preferablybound to magnetic beads, can be added. The tube can be rocked for aperiod of time for mixing the sample, and then positioned next to one ormore magnets for the capture of the magnetic beads. In this way, in asingle incubation and separation step, approximately 99% of RBCs and 99%of WBCs can be removed from a sample. The supernatant can be removedfrom the tube and subjected to filtration using a microfabricated filterof the present invention. Filtration removes remaining RBCs, resultingin a sample in which rare cells, such as, for example, fetal cells,cancer cells, candidate cancer cells, non-hematopoietic cells, or stemcells, have been enriched.

Undesirable components of a sample can be removed by methods other thanthose using specific binding members. For example, the dielectricalproperties of particular cell types can be exploited to separateundesirable components dielectrophoretically. For example, FIG. 22depicts white blood cells of a diluted blood sample retained onelectrodes of a dielectrophoresis chip after red blood cells have beenwashed through the chamber.

Combined Solution for Sedimenting Red Blood Cells and SelectivelyRemoving Undesirable Sample Components of a Blood Sample

In preferred embodiments of the present invention, a solution thatsediments red blood cells can also include one or more additionalspecific binding members that can be used to selectively removeundesirable sample components other than red blood cells from the bloodsample. In this regard, the present invention includes a combinedsedimenting solution for enriching rare cells of a blood sample thatsediments red blood cells and provides reagents for the removal of otherundesirable components of the sample. Thus a combined solution forprocessing a blood sample comprises: dextran; at least one specificbinding member that can induce agglutination of red blood cells; and atleast one additional specific binding member that can specifically bindundesirable components of the sample other than RBCs.

Specific Binding Member for Removing Undesirable Components

In addition to the components of a sedimenting solution of the presentinvention, a combined solution of the present invention can comprise atleast one specific binding member that can selectively bind undesirablecomponents of a blood sample (such as but not limited to white bloodcells, platelets, serum proteins) and have less binding to desirablecomponents. One or more specific binding members that can selectivelybind non-RBC undesirable components of a blood sample can be used toremove the undesirable components of the sample, increasing the relativeproportion of rare cells in the sample, and thus contribute to theenrichment of rare cells of the sample. By “selectively binds” is meantthat a specific binding member used in the methods of the presentinvention to remove one or more undesirable sample components does notappreciably bind to rare cells of interest of the fluid sample. By “doesnot appreciably bind” is meant that not more than 30%, preferably notmore than 20%, more preferably not more than 10%, and yet morepreferably not more than 1.0% of one or more rare cells of interest arebound by the specific binding member used to remove non-RBC undesirablecomponents from the fluid sample. In many cases, the undesirablecomponents of a blood sample will be white blood cells. In preferredembodiments of the present invention, a combined solution of the presentinvention can be used for sedimenting red blood cells and selectivelyremoving white blood cells from a blood sample.

A specific binding member that can specifically bind white blood cellscan be as nonlimiting examples, an antibody, a ligand for a receptor,transporter, channel or other moiety of the surface of a white bloodcell, or a lectin or other protein that can specifically bind particularcarbohydrate moieties on the surface of a white blood cell (for example,a selectin).

Preferably, a specific binding member that selectively binds white bloodcells is an antibody that binds white blood cells but does notappreciably bind fetal nucleated cells, such as, for example, anantibody to CD3, CD11b, CD14, CD17, CD31, CD45, CD50, CD53, CD63, CD69,CD81, CD84, CD102, or CD166. Antibodies can be purchased commerciallyfrom suppliers such as, for example Dako, BD Pharmingen, AntigenixAmerica, Neomarkers, Leinco Technologies, Research & Diagnostic Systems,Serotec, United States Biological, Bender Medsystems Diagnostics,Ancell, Leinco Technologies, Cortex Biochem, CalTag, Biodesign, Biomeda,Accurate Chemicals & Scientific and Chemicon International. Antibodiescan be tested for their ability to bind an efficiently remove whiteblood cells and allow for the enrichment of rare cells of interest froma sample using capture assays well known in the art.

Specific binding members that selectively bind to one or moreundesirable components of the present invention can be used to captureone or more non-RBC undesirable components, such that one or moredesirable components of the fluid sample can be removed from the area orvessel where the undesirable components are bound. In this way, theundesirable components can be separated from other components of thesample that include the rare cells to be separated. The capture can beaffected by attaching the specific binding members that recognize theundesirable component or components to a solid support, or by bindingsecondary specific binding members that recognize the specific bindingmembers that bind the undesirable component or components, to a solidsupport, such that the undesirable components become attached to thesolid support. In preferred embodiments of the present invention,specific binding members that selectively bind undesirable samplecomponents provided in a combined solution of the present invention arecoupled to a solid support, such as microparticles, but this is not arequirement of the present invention.

Magnetic beads are preferred solid supports for use in the methods ofthe present invention to which specific binding members that selectivelybind undesirable sample components can be coupled. Magnetic beads areknown in the art, and are available commercially. Methods of couplingmolecules, including proteins such as antibodies and lectins, tomicroparticles such as magnetic beads are known in the art. Preferredmagnetic beads of the present invention are from 0.02 to 20 microns indiameter, preferably from 0.05 to 10 microns in diameter, and morepreferably from 0.05 to 5 microns in diameter, and even more preferablyfrom 0.05 to 3 microns in diameter and are preferably provided in acombined solution of the present invention coated with a primaryspecific binding member, such as an antibody that can bind a cell thatis to be removed from the sample, or a secondary specific bindingmember, such as streptavidin, that can bind primary specific bindingmembers that bind undesirable sample components (such as biotinylatedprimary specific binding members).

In preferred embodiments of the present invention, the fluid sample is amaternal blood sample, the rare cells whose separation is desirable arefetal cells, and the undesirable components of the sample to be removedfrom the sample are white blood cells. In these embodiments, a specificbinding member that selectively binds white blood cells is used toremove the white blood cells from the sample by magnetic capture.Preferably, the specific binding member provided is attached to magneticbeads for direct capture, or, is provided in biotinylated form forindirect capture of white blood cells by streptavidin-coated magneticbeads.

A combined solution for enriching rare cells of a blood sample of thepresent invention can also include other components, such as, but notlimited to, salts, buffering agents, agents for maintaining a particularosmolality, chelators, proteins, lipids, small molecules,anticoagulants, etc. For example, in some preferred aspects of thepresent invention, a combined solution comprises physiological saltsolutions, such as PBS, PBS lacking calcium and magnesium or Hank'sbalanced salt solution. In some preferred aspects of the presentinvention, EDTA or heparin are present to prevent red blood cellclotting.

IV. Method of Enriching Rare Cells of a Blood Sample Using a Solutionthat Selectively Sediments Red Blood Cells

The present invention also includes a method of enriching rare cells ofa blood sample (including but not limited to e.g. peripheral blood)using a solution that selectively sediments red blood cells. The methodincludes: adding a red blood cell sedimenting solution of the presentinvention to a blood sample, mixing the blood sample and the red bloodcell sedimenting solution, allowing red blood cells to sediment from thesample, and removing a supernatant that comprises enriched rare cells.

Blood Sample

A blood sample can be any blood sample, recently taken from a subject,taken from storage, or removed from a source external to a subject, suchas clothing, upholstery, tools, etc. A blood sample can therefore be anextract obtained, for example, by soaking an article containing blood ina buffer or solution. A blood sample can be unprocessed or partiallyprocessed, for example, a blood sample that has been dialyzed, hadreagents added to it, etc. In some cases, it can be preferable to use awashed blood sample, in which blood cells have been pelleted andresuspended in a blood-compatible buffer (for example, PBE) at leastonce. A blood sample can be of any volume. For example, a blood samplecan be less than five microliters, or more than 5 liters, depending onthe application. Preferably, however, a blood sample that is processedusing the methods of the present invention will be from about 10microliters to about 2 liters in volume, more preferably from about onemilliliter to about 250 milliliters in volume, and most preferablybetween about 5 and 50 milliliters in volume.

Addition of Sedimenting Solution to Sample

A red blood cell sedimenting solution can be added to a blood sample byany convenient means, such as pipetting, automatic liquiduptake/dispensing devices or systems, pumping through conduits, etc. Inmost cases, the blood sample will be in a tube that provides for optimalseparation of sedimented cells, but it can be in any type of vessel forholding a liquid sample, such as a plate, dish, well, or chamber. Theamount of sedimenting solution that is added to a blood sample can vary,and will largely be determined by the concentration of dextran andspecific binding members in the sedimenting solution (as well as othercomponents), so that their concentrations will be optimal when mixedwith the blood sample. Optimally, the volume of a blood sample isassessed, and an appropriate proportional volume of sedimentingsolution, ranging from 0.01 to 100 times the sample volume, preferablyranging from 0.1 times to 10 times the sample volume, and morepreferably from 0.25 to 5 times the sample volume, and even morepreferably from 0.5 times to 2 times the sample volume, is added to theblood sample. (It is also possible to add a blood sample, or a portionthereof, to a red blood cell sedimenting solution. In this case, a knownvolume of sedimenting solution can be provided in a tube or othervessel, and a measured volume of a blood sample can be added to thesedimenting solution.)

Mixing

The blood sample and red blood cell sedimenting solution are mixed sothat the chemical RBC aggregating agent (such as a polymer, such as, forexample, dextran) and one or more specific binding members of thesedimenting solution, as well as the components of the blood sample aredistributed throughout the sample vessel. Mixing can be achieved meanssuch as electrically powered acoustic mixing, stirring, rocking,inversion, agitation, etc., with methods such as rocking and inversion,that are least likely to disrupt cells, being favored.

Incubation of Blood Sample and Sedimenting Solution

The sample mixed with sedimenting solution is allowed to incubate toallow red blood cells to sediment. Preferably the vessel comprising thesample is stationary during the sedimentation period so that the cellscan settle efficiently. Sedimentation can be performed at anytemperature from about 5 degrees C. to about 37 degrees C. In mostcases, it is convenient to perform the steps of the method from about 15degrees C. to about 27 degrees C. The optimal time for the sedimentationincubation can be determined empirically for a given sedimentingsolution, while varying such parameters as the concentration of dextranand specific binding members in the solution, the dilution factor of theblood sample after adding the sedimenting solution, and the temperatureof incubation. Preferably, the sedimentation incubation is from fiveminutes to twenty four hours in length, more preferably from ten minutesto four hours in length, and most preferably from about fifteen minutesto about one hour in length. In some preferred aspects of the presentinvention, the incubation period is about thirty minutes.

Collecting Enriched Cells

Removing a supernatant (or a portion thereof) from the sample after thered blood cells have sedimented can be performed by pouring, pipetting,pumping, or a fluid uptake device. The supernatant comprises enrichedrare cells of the blood sample, such as, but not limited to, stem cells,fetal cells, nucleated red blood cells, mesenchymal cells,subpopulations of blood cells (including but not limited to e.g. Tcells, dendritic cells), non-hematopoietic cells (including but notlimited to e.g. epithelial cells, endothelial cells), cancer cells,candidate cancer cells, pre-neoplastic cells, neoplastic cells,metastatic cells, virus-infected cells, parasite-infected cells,parasitic cells, or bacterial cells. Following RBC sedimentation with aRBC sedimenting solution of the present invention, the proportion of therare cells to the other cell types in the sample has increased, thusresulting in enriched rare cells.

Method of Enriching Rare Cells of a Blood Sample Using a CombinedSedimenting Solution

The present invention also includes a method of enriching rare cells ofa blood sample using a combined solution for enriching rare cells of ablood sample. The method comprises: adding a combined solution forenriching rare cells of the present invention to a blood sample in atube or vessel; mixing the blood sample and combined solution of thepresent invention; allowing red blood cells to sediment from the bloodsample; allowing undesirable components to bind a solid support; andremoving a supernatant from said blood sample that comprises enrichedrare cells.

Blood Sample

A blood sample can be any blood sample, recently taken from a subject,taken from storage, or removed from a source external to a subject, suchas clothing, upholstery, tools, etc. A blood sample can therefore be anextract obtained, for example, by soaking an article containing blood ina buffer or solution. A blood sample can be unprocessed or partiallyprocessed, for example, a blood sample that has been dialyzed, hadreagents added to it, etc. In some cases, it can be preferably to use awashed blood sample, in which blood cells have been pelleted andresuspended in a blood-compatible buffer (for example, PBE) at leastonce. A blood sample can be of any volume. For example, a blood samplecan be less than five microliters, or more than 5 liters, depending onthe application. Preferably, however, a blood sample that is processedusing the methods of the present invention will be from about 10microliters to about 2 liters in volume, more preferably from about onemilliliter to about 250 milliliters in volume, and most preferablybetween about 5 and 50 milliliters in volume.

Addition of Sedimenting Solution to Sample

A combined sedimenting solution can be added to a blood sample by anyconvenient means, such as pipetting, automatic liquid uptake/dispensingdevices or systems, pumping through conduits, etc. In most cases, theblood sample will be in a tube that provides for optimal separation ofsedimented cells, but it can be in any type of vessel for holding aliquid sample, such as a plate, dish, well, or chamber. The amount ofcombined sedimenting solution that is added to a blood sample can vary,and will largely be determined by the concentration of dextran andspecific binding members in the combined solution (as well as othercomponents), so that their concentrations will be optimal when mixedwith the blood sample. Optimally, the volume of a blood sample isassessed, and an appropriate proportional volume of combined solution,preferably ranging from 0.1 times to 10 times the sample volume, andmore preferably from 0.25 to 5 times the sample volume, and even morepreferably from 0.5 times to 2 times the sample volume, is added to theblood sample. (It is also possible to add a blood sample, or a portionthereof, to a combined solution. In this case, a known volume ofcombined solution can be provided in a tube or other vessel, and ameasured volume of a blood sample can be added to the combinedsolution.)

Mixing

The blood sample and combined sedimenting solution are mixed so that thedextran and specific binding members of the combined solution, as wellas the components of the blood sample, are distributed throughout thesample vessel, and specific binding members can bind to samplecomponents. Mixing can be achieved means such as electrically poweredacoustic mixing, stirring, rocking, inversion, agitation, etc., withmethods such as rocking and inversion, that are least likely to disruptcells, being favored.

Incubation of Blood Sample and Combined Solution

The sample mixed with combined sedimenting solution is allowed toincubate to allow red blood cells to sediment. Preferably the vesselcomprising the sample is stationary during the sedimentation period sothat the red blood cells can settle efficiently. Sedimentation can beperformed at any temperature from about 5 degrees C. to about 37 degreesC. In most cases, it is convenient to perform the steps of the methodfrom about 15 degrees C. to about 27 degrees C. The optimal time for thesedimentation incubation can be determined empirically for a givencombined sedimenting solution, while varying such parameters as theconcentration of dextran and specific binding members in the solution,the dilution factor of the blood sample after adding the combinedsolution, and the temperature of incubation. Preferably, thesedimentation incubation is from ten minutes to twenty four hours inlength, more preferably from fifteen minutes to one hour in length. Insome preferred aspects of the present invention, the incubation periodis about thirty minutes.

Allowing Undesirable Sample Components or Rare Cells Bound by SpecificBinding Members to Bind a Solid Support

Allowing undesirable components or rare cells bound by specific bindingmembers to bind a solid support can be performed in any of several ways,depending on the nature of the specific binding member that bindsundesirable sample components, the type of solid support, and theoverall format of the enrichments procedure (whether it is performed inone or more vessels, whether fluid flow is involved, etc.). In someembodiments, after the sedimentation step, the supernatant can be passedthrough or over a solid support that comprises secondary specificbinding members that can bind the primary specific binding members (forexample, streptavidin, if the primary specific binding member isbiotinylated). For example, the supernatant can be pipetted or pumpedthrough a column or over a membrane that can capture the undesirablecomponents or rare cells bound by specific binding members. In otherembodiments, one or more specific binding members that can bindundesirable sample components or rare cells can be bound to a solidsupport, such as beads, that can be sedimented along with the red bloodcells, with or without a centrifugation step.

In preferred embodiments of the present invention, magnetic beads aresolid supports, and one or more specific binding members that bindundesirable sample components are bound to magnetic beads in a combinedsedimenting solution of the present invention. The magnetic beads can becaptured using a magnet before, during or after the sedimentation step.In preferred aspects of the present invention, during the sedimentationstep magnetic beads comprising primary or secondary specific bindingmembers for the capture of undesirable components or rare cells of theblood sample are collected by placing the vessel that contains thesample next to a magnet. Magnetic capture can also be performed when thecombined solution comprises a specific binding member that canspecifically bind undesirable components or rare cells or interest canbe bound by magnetic beads that are coated with, for example,streptavidin (where the specific binding member is biotinylated).

Preferably, magnetic capture uses one or more permanent magnets, such aspermanent magnets positioned within or alongside a tube, dish, or vesselthat contains the target cell-magnetic bead complexes, and occurs duringthe sedimentation step. Commercially available magnetic separators thatinclude permanent magnets (such as those sold by Immunicon (HuntingtonValley, Pa.)) can also be used, however, or magnetic capture can alsoemploy electromagnetic chips that comprise electromagnetic physicalforce-generating elements, such as those described in U.S. Pat. No.6,355,491 entitled “Individually Addressable Micro-Electromagnetic UnitArray Chips” issued Mar. 12, 2002 to Zhou et al., U.S. application Ser.No. 09/955,343 having attorney docket number ART-00104.P.2, filed Sep.18, 2001, entitled “Individually Addressable Micro-Electromagnetic UnitArray Chips”, and U.S. application Ser. No. 09/685,410 having attorneydocket number ART-00104.P.1.1, filed Oct. 10, 2000, entitled“Individually Addressable Micro-Electromagnetic Unit Array Chips inHorizontal Configurations”.

In preferred embodiments, combined solution of the present inventioncomprises at least one specific binding member that selectively bindswhite blood cells as undesirable components of the sample. The specificbinding member is bound to, or is able to bind to, magnetic beads. Thetube containing the sample mixed with the combined solution ispositioned next to a magnet during sedimentation of red blood cells, andwhite blood cells are collected at the wall of the tube as red bloodcells settle to the bottom of the tube. The supernatant comprisesenriched rare cells.

Collecting Enriched Rare Cells

The process of collecting enriched cells will vary depending on whethera combined sedimenting solution comprises a specific binding member thatselectively binds undesirable sample components or a specific bindingmember that selectively binds rare cells of interest. In embodiments inwhich a combined sedimenting solution comprises a specific bindingmember that selectively binds undesirable sample components, removing asupernatant (or a portion thereof) from the sample after the red bloodcells have sedimented and undesirable sample components have beenseparated can be performed by pouring, pipetting, pumping, or a fluiduptake device. The supernatant comprises enriched rare cells of theblood sample, such as, but not limited to, stem cells, fetal cells,nucleated red blood cells, cancer cells, candidate cancer cells,virus-infected cells, parasite-infected cells, parasitic cells, orbacterial cells. The proportion of these cells relative to the totalcell population in the collected supernatant has increased over theirproportion of the total cell population in the pre-sedimented bloodsample.

Further Enrichment Steps

The use of sedimenting solutions of the present invention, includingcombined solutions for enriching rare cells of a blood sample, can becombined with other processing steps such as debulking, or separationsteps. Debulking steps that can be combined with the use of a combinedsolution include: as nonlimiting examples, selective lysis, filtration,and centrifugation steps. Additional separation steps that can be usedinclude separations that include capture of sample components to solidsupports using specific binding members, and separations performed onactive chips, such as dielectrophoretic and traveling wavedielectrophoretic separations, and separations using electromagneticcapture on an electromagnetic chip.

The present invention also includes methods of enriching rare cells froma blood sample in which selective sedimentation of RBCs is combined withfiltration, such as filtration through a microfabricated filter of thepresent invention.

A method for enriching rare cells of the present invention thatcomprises a RBC sedimentation step and at least one filtration stepusing a microfabricated filter of the present invention can also includeother steps, such as, but not limited to: selectively removing furtherundesirable components from said fluid sample, separating rare cells ofthe sample, additional filtration steps, or additional debulking steps,such as, for example, selective lysis of one or more sample components.

In a particularly preferred embodiment, a blood sample can be processedto enrich rare cells, which may include but are not limited to fetalcells, stem cells, cancer cells or candidate cancer cells, ornon-hematopoietic cells. Red blood cells can be removed by selectivesedimentation of RBCs using a solution of the present invention. Whiteblood cells can be removed by adding magnetic beads that are coated withone or more specific binding members that specifically bind white bloodcells to the post-sedimentation supernatant, or, preferably, a combinedsolution of the present invention is used to sediment red blood cellsand removes white blood cells using magnetic capture. The blood samplecan then be dispensed into a filtration chamber that comprises at leastone microfabricated filter of the present invention that comprises slotshaving dimension that allow RBCs to pass through the filter. Fluid flowthrough the chamber removes additional residual red blood cells andfurther reduces sample volume, resulting in a sample having enrichedrare cells. Depending on the source of the sample, the enriched rarecells can be stem cells, fetal cells, cancer cells, candidate cancercells, subtypes of white blood cells, bacterial cells, parasite cells,or bacteria-, parasite-, or virus-infected cells.

Additional Debulking Steps

As used herein, “debulking” refers to a step in the processing of asample in which the volume of the sample is significantly reduced by atleast fifty per cent or greater than 50% of the cellular components of asample are removed. For example, in preferred aspects of the presentinvention in which the fluid sample is a blood sample, a majority of thenon-nucleated red blood cells (RBCs) that make up more than 90% of thecellular components of a blood sample are removed during a debulkingstep.

Additional debulking steps used before or after sedimenting red bloodcells with a solution of the present invention can be, as nonlimitingexamples, an additional sedimentation step, a concentration step, acentrifugation step, or a filtration step. Centrifugation and filtrationare preferred debulking steps that reduce the volume of a fluid sampleand at the same time allow the technician to select fractions of thecentrifuged or filtered product that retain desirable components and donot retain at least a portion of some undesirable components.

Filtration using a microfabricated filter of the present invention hasbeen disclosed earlier in the application. Other types of filtrationsteps can also be used. These include, as nonlimiting examples,filtration using columns packed with various resins or polymericmaterials, filtration using membranes of pore sizes that allow retentionof desirable components, filtration using channels that are microetchedinto one or more chips, by using “bricks” or dams that are built ontothe surface of a chip, or by using slots or pores that are microetchedinto a solid surface that can be within a chamber or form a wall of achamber as disclosed earlier in the application (see, for example, U.S.Pat. No. 5,837,115 issued Nov. 17, 1998 to Austin et al., hereinincorporated by reference in its entirety and U.S. Pat. No. 5,726,026issued Mar. 10, 1998 to Wilding et al., herein incorporated by referencein its entirety).

Another method for debulking blood sample is the use of hypotonicsolutions to exploit the differential responses of maternal red bloodcells (and reticulocytes) and white blood cells (and nucleated red bloodcells). By treating blood samples with hypotonic solutions, red bloodcells can be lysed, or red blood cells can be altered significantly sothat they become readily separable from white blood cells and othernucleated cells. Alternatively, certain biochemical reagents may be usedto selectively lyse red blood cells.

More than one debulking step can be employed in the methods of thepresent invention. For example, in some applications, undesirablecomponents of the sample can be removed in steps subsequent to a firstdebulking step. It can then practical and advantageous to further reducethe volume of the remaining sample. This can be done through any of thedescribed debulking methods, using scaled down volumes and areas whereappropriate.

Separation Steps

The methods of the present invention can include sedimentation of redblood cells from a blood sample in combination with one or moreseparation steps. In general, a separation step will selectively removeone or more undesirable components from a sample, or selectivelyseparate one or more desirable components of a sample. These steps willdepend on the properties of the particular cells to be removed orseparated from the sample, such as their binding properties, physicalproperties such as size or density, and electrical properties.

Sedimenting RBCs with Selectively Removal of Undesirable Components andFurther Selective Removal of Undesirable Components.

The present invention includes methods in which the selective removal ofone or more non-RBC undesirable components of a fluid sample isperformed simultaneously with sedimenting red blood cells of a sample.However, in some methods of the present invention, in which asedimenting solution does not comprise a specific binding member thatselectively binds non-RBC undesirable components, removal of one or moreundesirable components of a fluid sample can be performed before,during, or after sedimenting red blood cells from the blood sample. Itis also possible to remove more than one type of undesirable componentfrom a blood sample, and to perform the separations in separate steps.

Preferably, in the methods of the present invention, selective removalof one or more undesirable components of a fluid sample makes use ofspecific recognition of one or more undesirable components by one ormore specific binding members. The use of specific binding members inremoving undesirable components of a sample has been disclosed inearlier sections of the application and also applies here. The specificbinding member used in the methods of the present invention can be anytype of molecule or substrate that can specifically bind one or moreundesirable components. Receptor ligands (either naturally occurring,modified, or synthetic), antibodies, and lectins are nonlimitingexamples of specific binding members that can be used in the methods ofthe present invention. More than one different specific binding membercan be used to capture one or more undesirable components to a solidsupport. Preferably, a specific binding member used in the methods ofthe present invention to selectively remove one or more undesirablecomponents does not appreciably bind to desirable components of thefluid sample. In most applications of the present invention, a specificbinding member used in the methods of the present invention to removeone or more undesirable components does not appreciably bind to the rarecells of the fluid sample that are to be separated. By “does notappreciably bind” is meant that not more than 30%, preferably not morethan 20%, more preferably not more than 10%, and yet more preferably notmore than 1.0% of the rare cell of the fluid sample that are to beenriched using the methods of the present invention are bound by thespecific binding member used to selectively remove undesirablecomponents of the fluid sample. Preferred specific binding members usedin the methods of the present invention include antibodies, particularlyantibodies that recognize and bind cell surface epitopes.

The capture can be affected by attaching antibodies that recognize theundesirable component or components to a solid support, or by bindingsecondary specific binding members that recognize the antibodies thatbind the undesirable component or components, to a solid support, suchthat the undesirable components become attached to the solid support andbecome fixed at a particular location. A solid support can be, asnonlimiting examples, a surface, such as a plastic or polymeric surface,a gel or polymer, a membrane, the surface of a chip, or a bead. In thepresent invention, magnetic beads are preferred solid supports for thecapture and selective removal of undesirable components of a sample. Thecapture of undesirable components of a sample can be direct or indirect.For direct capture, a first specific binding member that binds to one ormore undesirable components of a sample can be attached to a solidsupport. The one or more undesirable components, when contacted with thesolid support, then bind to the solid support. For indirect capture, aprimary specific binding member that binds to one or more undesirablecomponents of a sample can be contacted with the one or more undesirablecomponents, and a secondary specific binding member that can bind theprimary specific binding member can be attached to a solid support. Whenthe undesirable components that have bound the primary specific bindingmember are contacted with the solid support, the one or more undesirablecomponents of the sample can bind the solid support via the primary andsecondary specific binding members. In certain preferred embodiments ofthe present invention where selective removal of one or more undesirablecomponents of a sample is performed, direct capture is preferred, asdirect capture comprises fewer steps.

The capture of undesirable components of a sample could also be doneusing specific binding members that recognize cell surface antigens orrecognize the antibodies that bind the cell surface antigens and removedby other manipulations. An example but not limited to would be anantibody that recognizes an undesirable cell surface antigen, where theantibody could be labeled but are not limited to with markers that arechromophore, fluorescent, emit a signal, nanocrystal, microparticle,colloid, metal particle or other detectable reagent. This labeling ofundesired components can be utilized before, during or after either thedebulking or separation step. An example but not limited to utilizationcould be after the filtration step where the labeled undesirablecomponents are then flow cytometry sorted for separation of unlabeledcells and thus enrichment by further removal of undesirable components.

In preferred embodiments of the present invention, the fluid sample is amaternal blood sample, the rare cells whose separation is desirable arefetal cells, and the undesirable components of the sample to be removedfrom the sample are white blood cells. In these embodiments, a specificbinding member that selectively binds white blood cells is used toremove the white blood cells from the sample by magnetic capture.Preferably, the specific binding member is either used to coat magneticbeads for direct capture, or is used in biotinylated form for indirectcapture of white blood cells by streptavidin-coated magnetic beads.

A blood sample from which red blood cells have been sedimented can beincubated with one or more specific binding members, such as, but notlimited to, antibodies, that specifically recognize one or moreundesirable components of a fluid sample. Mixing and incubation of oneor more specific binding members with the sample can be performed in atube, dish, vessel, or chamber. The one or more undesirable componentscan be captured, either directly or indirectly, via their binding to thespecific binding member. For example, a specific binding member can bebound to a solid support, such as a bead, membrane, or column matrix,and following incubation of the fluid sample with the specific bindingmember, the fluid sample, containing unbound components, can be removedfrom the solid support. Alternatively, one or more primary specificbinding members can be incubated with the fluid sample, and the fluidsample can be contacted with a secondary specific binding member thatcan bind or is bound to a solid support. In this way the one or moreundesirable components of the sample can become bound to a solidsupport, enabling separation of the undesirable components from thefluid sample.

In one preferred embodiment, after incubation of magnetic beads thatcomprise a specific binding member that specifically bind undesirablecomponents with a sample, the sample is transported through a separationcolumn that comprises or engages at least one magnet. As the sampleflows through the column, undesirable components that are bound to themagnetic beads adhere to one or more walls of the tube adjacent to themagnet or magnets. An alternative embodiment uses a magnetic separator,such as the magnetic separator manufactured by Immunicon. Magneticcapture can also employ electromagnetic chips that compriseelectromagnetic physical force-generating elements, such as thosedescribed in U.S. Pat. No. 6,355,491 entitled “Individually AddressableMicro-Electromagnetic Unit Array Chips” issued Mar. 12, 2002 to Zhou etal., U.S. application Ser. No. 09/955,343 having attorney docket numberART-00104.P.2, filed Sep. 18, 2001, entitled “Individually AddressableMicro-Electromagnetic Unit Array Chips”, and U.S. application Ser. No.09/685,410 having attorney docket number ART-00104.P.1.1, filed Oct. 10,2000, entitled “Individually Addressable Micro-Electromagnetic UnitArray Chips in Horizontal Configurations”. In yet another preferredembodiment, a tube that contains the sample and magnetic beads ispositioned next to one or more magnets for the capture of undesirablecomponents bound to magnetic beads. The supernatant, depleted of the oneor more undesirable components, can be removed from the tube after thebeads have collected at the tube wall.

Other manipulations that can be performed to remove undesirablecomponents from a blood sample before or preferably after sedimentingred blood cells include passing the sample or sample supernatant over asolid support (which can be, as nonlimiting examples, a membrane or amatrix) that comprises attached specific binding members that capturethe undesirable components. The blood sample Or blood sample supernatantcan be incubated with or passed through or over such a solid support toremove undesirable components, such as, but not limited to, white bloodcells. Flow cytometry, dielectrophoretic separation, filtration, orother separation techniques can also optionally be employed to removeundesirable components from blood samples.

Sedimenting RBCs with Selective Removal of Undesirable Components andFurther Separating Desirable Components

The present invention also includes methods in which sedimenting rarecells is combined with the separation of one or more desirablecomponents, such as rare cells whose enrichment is desired, from a fluidsample. Preferably, separation of rare cells from a fluid sample occursafter red blood cell sedimentation.

In some preferred embodiments of the present invention, separating rarecells uses at least one specific binding member that specifically bindsthe one or more rare cells and capture of the rare cells to a solidsupport. Receptor ligands (either of natural sources, modified, orsynthetic), antibodies, and lectins are nonlimiting examples of specificbinding members that can be used in the methods of the presentinvention. More than one different specific binding member can be usedto capture one or more rare cells to a solid support.

A specific binding member can be any type of molecule or substrate thatcan selectively bind one or more rare cell types. Preferred specificbinding members used in the methods of the present invention includeantibodies, particularly antibodies that recognize and bind antigens onthe surface of rare cells.

In a particularly preferred embodiment, the fluid sample is a bloodsample and fetal nucleated cells are the rare cells to be enriched. Inthis case, specific binding members such as lectins or antibodies can beused to bind and remove white blood cells.

Antibodies can also be used as specific binding members for othermanipulations such as to label fetal nucleated cells from a bloodsample. For example, a CD71 antibody can be used. An antibody orantibodies can also be used to enrich other rare cells such as, forexample, cancer cells or stem cells from fluid samples such as urine orblood samples. Antibodies, lectins, or other specific binding memberscan be tested for their ability to bind an efficiently separateparticular rare cell types from a sample using capture assays well knownin the art.

A blood sample from which red blood cell have been sedimented can beincubated with one or more specific binding members, such as antibodies,that specifically recognize one or more rare cell types of a fluidsample. The one or more rare cell types can be captured, via theirdirect or indirect binding to the specific binding member, and theremainder of the fluid sample can be removed from the area, surface, orvessel where the rare cells being isolated are bound. For example, aspecific binding member can be bound to a solid support, such as amembrane or column matrix, and following incubation of the fluid samplewith the specific binding member, the fluid sample, containing unboundcomponents, can be removed from the solid support. A solid support canbe, as nonlimiting examples, a surface, such as a plastic surface, a gelor polymer, a membrane, the surface of a chip, or a bead. In the presentinvention, magnetic beads are preferred solid supports for theseparation and capture of rare cells of a sample.

Capture of cells, viruses, molecules, and other moieties to solidsupports is well known in the arts of cell biology, biochemistry, andantibody technology, and can use a variety of formats known in the art.The capture of rare cells of a sample can be direct or indirect. Fordirect capture, a first specific binding member that binds to one ormore rare cells of a sample can be attached to a solid support. The rarecells, when contacted with the solid support, then bind to the solidsupport. For indirect capture, a primary specific binding member thatbinds to the desirable rare cells of a sample can be contacted with theone or more rare cells, and a secondary specific binding member that canbind the primary specific binding member can be attached to a solidsupport. When the rare cells that have bound the primary specificbinding member are contacted with the solid support, the one or morerare cells of the sample can bind the solid support via the primary andsecondary specific binding members.

In many cases it can be preferable to provide the specific bindingmember that binds the rare cells already bound to a solid support. Forexample, beads, such as magnetic beads, to which one or more specificbinding members that bind the rare cells are attached can be added tothe sample, or the sample can be passed over a solid support such as amembrane or the surface of a plate that comprises a specific bindingmember, or through a solid support such as a column matrix thatcomprises a specific binding member. Using specific binding members thatare directly bound to a solid support can increase the efficiency of theenrichment procedure.

In preferred embodiments, separation of one or more rare cells of thesample using specific binding members to capture the rare cells to asolid support, and can be performed in a dish, well, tube, column, orother vessel. In some preferred embodiments, the solid support comprisesmagnetic beads.

Magnetic beads are preferred solid supports for use in the methods ofthe present invention. Magnetic beads are known in the art, and areavailable commercially. Magnetic beads can be purchased that are coatedwith secondary specific binding members, for example secondaryantibodies or streptavidin. Preferred magnetic beads of the presentinvention are from 0.02 to 20 microns in diameter, preferably from 0.05to 10 microns in diameter, and more preferably from 0.05 to 5 microns indiameter, and even more preferably from 0.05 to 3 microns in diameterand are coated with either streptavidin, a secondary antibody, or aprimary antibody that can bind a cell that is to separated from thesample. Where streptavidin coated beads are used, the primary specificbinding member is preferably biotinylated (for example a biotinylatedprimary antibody) such that the streptavidin coated bead will bind asample component that is bound to the biotinylated antibody through astreptavidin-biotin link. Methods of using magnetic beads in the captureof directly or indirectly bound cells are well known in the art, and arealso described in the examples provided. The methods of capture can usepermanent magnets, such as permanent magnets positioned within oralongside a tube, dish, or vessel that contains the target cell-magneticbead complexes, or commercially available magnetic separators thatinclude permanent magnets (Immunicon). Magnetic capture can also employelectromagnetic chips that comprise electromagnetic physicalforce-generating elements, such as those described in U.S. Pat. No.6,355,491 entitled “Individually Addressable Micro-Electromagnetic UnitArray Chips” issued Mar. 12, 2002 to Zhou et al., U.S. application Ser.No. 09/955,343 having attorney docket number ART-00104.P.2, filed Sep.18, 2001, entitled “Individually Addressable Micro-Electromagnetic UnitArray Chips”, and U.S. application Ser. No. 09/685,410 having attorneydocket number ART-00104.P.1.1, filed Oct. 10, 2000, entitled“Individually Addressable Micro-Electromagnetic Unit Array Chips inHorizontal Configurations”.

A discussion and references of the use of electromagnetic forces andtheir use is separations provided in a previous section of thisapplication on methods of enriching rare cells involving filtration canalso be applied to the separation of rare cells following RBCsedimentation.

Rare cells of the present invention can also be separated from a fluidsample using dielectrophoretic forces. The use of dielectrophoreticforces can be employed where the rare target cells havedielectrophoretic properties than are significantly different than othercomponents that remain in the sample. That is, the difference indielectrophoretic properties between rare target cells and nondesirablesample components must be sufficient to allow dielectrophoreticseparation using micro-scale electrodes that can be built into or onto achip. In most cases in which the fluid sample is a biological fluidsample, the other components of the sample whose dielectric propertiesmust be taken into account are cells, such as cells that are not raretarget cells. The feasibility of using dielectrophoresis for theseparation of rare target cells can therefore depend on whethernondesirable components having similar dielectrophoretic properties asthe target cells. Preferably, then, in applications of the method wherea sample comprises a type of non-target cells that have similardielectrophoretic properties as the target cells, selective removal ofthe type of non-target cells using methods other than dielectrophoresishas been performed prior to dielectrophoretic separation of targetcells. Preferably in such instances, the selective removal of thenon-target cells with similar dielectric properties using methods otherthan dielectrophoresis has been efficient, where efficiency refers tothe percentage of non-target cells removed. The level of efficiency canvary with the application, but preferably the efficiency of selectiveremoval of non-target cells with similar dielectric properties isgreater than 30% of the non-target cells removed, more preferablygreater than 50% of the non-target cells removed, and more preferablyyet, greater than 90% of the non-target cells removed, and even morepreferably, greater than 99% of the non-target cells removed in theselective removal step.

The previous discussion and references provided for the design and useof micro-electrodes to facilitate filtration by translocating samplecomponents, such as nonfilterable cells, away from a filter usingdielectrophoresis are also relevant to the use of micro-electrodes tofacilitate dielectrophoretic separation of rare target cells. Variousdielectrophoresis separation methods, such as those described in U.S.application Ser. No. 09/686,737, filed Oct. 10, 2000 entitled“Compositions and Methods for Separation of Moieties on Chips”,incorporated by reference, and described in U.S. application Ser. No.09/679,024 having attorney docket number 471842000400, entitled“Apparatuses Containing Multiple Active Force Generating Elements andUses Thereof” filed Oct. 4, 2000, also herein incorporated by referencein its entirety, may be employed for separating rare target cells.

In some applications of the present invention, separation of rare cellsfrom a fluid sample may exploit the differences in cell physicalproperties. For example, as discussed above, dielectrophoresis may beused to separate nucleated red blood cells from non-nucleated red bloodcells By exploiting the differences in their dielectric properties,nucleated red blood cells and mature red blood cells (and reticulocytes)are caused to exhibit positive and negative (or small positive)dielectrophoresis forces, respectively, under certain cell suspensionand electric field conditions. When the cell suspension is introduced toa chamber containing microelectrodes on the bottom surface, nucleatedred blood cells can be collected and retained on the electrodes whilethe non-nucleated red blood cells are carried away from the chambertogether with the fluid stream.

Other manipulations that can be performed to separate rare cells from ablood sample before or preferably after sedimenting red blood cellsinclude passing the sample or sample supernatant over a solid support(which can be, as nonlimiting examples, a membrane or a matrix) thatcomprises attached specific binding members that capture the undesirablecomponents. The blood sample or blood sample supernatant can beincubated with or passed through or over such a solid support to collectthe rare cells.

In addition to the manipulations setforth above rare cells may beenriched by further methods of manipulation using methods of detection,identification, characterization, culture and the like. These methodsmay have particular utility when used in combination with debulking ofsamples such as blood samples.

Fluid Volume Sensing Means

An automated system of the present invention can have means for sensingthe volume of a fluid, such as, but not limited to, the volume of afluid sample, including a fluid sample supernatant. The means forsensing the volume of a fluid preferably relies on optical sensing, suchas detection of transmittance, absorption, reflectance, or fluorescence,and can comprise a light source, such as a light bulb, laser, or LED,and a sensing structure such as CCDs or photomultipliers appropriatelyaligned with the light source or sources. Thus the volume sensing meanscan comprise a light transmission-light sensing system that does notrely on contacting the sample to detect volume. Wavelengths forparticular sensing applications can be readily determined, for example,for turbidity (600 nm), or the absorbance of particular samplecomponents. A light source that is part of a light transmission-lightsensing system can transmit light in the non-visible range, such as theultraviolet or infrared range. For example, the fraction of a samplethat comprises red blood cells can be detected using light in the rangeof 700 to 900 nanometers, more preferably between 750 and 850nanometers.

In a preferred embodiment of fluid volume sensing means, the lightsource is a laser that emits collated light, that is, filtered,polarized light that can transmit through a sample tube, and in somepreferred embodiments, can transmit through a sample or a fraction of asample that does not absorb at the wavelength of the emitted light. (Thetube, vessel, or other container that holds the sample whose volume isto be determined should be transparent to, or substantially transparentto, the emitted light.)

A light source and detection device can be mobile, so that they cancontinuously or in graduated fashion scan the length of the tube orcolumn that contains the sample, or the fluid volume sensing means canhave multiple light sources and multiple detectors that are orientedvertically and can simultaneously detect optical parameters and therebydetermine the volume of a sample (or a subfraction thereof). Becauseblood samples contain cells such as RBCs and WBCs, a change in theoptical characteristics can determine the locus of particular celltypes. It is also possible to fluorescently label cells so thatfluorescence can be used for localization.

For example, a light source, such as but not limited to a light bulb,laser, or LED, can interrogate the tube or column of sample. Thetransmittance, absorption or reflectance of the incident light can bemeasured by appropriate structures, such as CCDs or photomultipliers.

The automated fluid volume sensing means can be used to determine thevolume of a sample or a portion thereof at any of various stages in theprocessing of a sample. In one embodiment, fluid volume sensing meanscan determine the starting volume of a sample by detecting, for example,absorbance/transmission of light of a given wavelength along the lengthof a tube, vial, cuvette, etc. This can be used, for example, tocalculate the amount of a reagent to add to a sample. In a preferredmethod for processing a blood sample, the automated system calculatesthe amount of combined solution to add to each sample tube, and adds theappropriate amounts using an automated fluid dispensing system.

In other embodiments, fluid volume sensing means can be used todetermine the volume of a fraction of a sample. For example, a sampleprecipitate can have different light absorption characteristics than asample supernatant, or two phases of a separated sample can havedifferent light absorption characteristics. In a preferred example, aninterface between sedimented red blood cells and a sample supernatantcan be localized using fluid volume sensing means.

Because layers of the sample column with a high density of RBCs areoptically dense and do not transmit light well, the interface betweenhigh and low RBC densities can be determined by such optical methods.The instrument can localize such interface or zone and calculate thevolume of a precipitate, supernatant, or phase of the sample.

In this sense, “calculate a volume” does not require that the systemperform a calculation that arrives at a volume per se, although this canbe done. In most embodiments, the automated system will determine aheight or level or a sample or interface or boundary, and thisdetermination will direct the fluid uptake system to remove a certainamount of sample or add a certain amount of reagent or solution to thesample.

This can be done by using a light source and detection device that aremobile, and either continuously or in graduated fashion scans the lengthof the tube or column, or by having multiple light sources and detectorsthat are oriented vertically and can simultaneously detect opticaldensity and thereby determine the volume of the sample (or subfractionthereof). Preferably, a light source moves in a coordinated fashion witha light detection device to scan a sample tube or vessel. In a preferredembodiment, a light transmission-light sensing device comprises a baroriented essentially horizontally and having on one end a light source,such as a collated light source, and on the other end, a light sensor.The bar is proximal to or can be positioned proximal to the sample tubein a rack, such that the light source is on one side of the tube, andthe light sensor is at the opposite end of the tube. The upper level ofthe bar corresponds to the level of the light source.

To determine the volume of a sample supernatant, the bar moves upwardfrom the level of the bottom of the tube. The detection device recordsthe amount of light through the sample, and, when a boundary is detected(light transmission reaches a threshold value or significant differencein the amount of transmitted light within a short distance), the barstops at the boundary position, for example, at the interface betweenfractions of a sample, such as a sample supernatant and a sampleprecipitate.

The boundary determination can be used to direct a fluiduptake/dispensing system to remove the upper phase or supernatant of thesample. The sample supernatant or upper phase can be removed bydirecting the tip of a fluid uptake device relative to the level of thedetected interface. This can be done by having the collection tipposition itself over the bar and move downward to the bar (which hasstopped at the level of the interface). When the tip contacts the bar,the tip level is recorded. The collection tip then moves back up,positions itself over the sample tube, and descends into the sampletube. When the tip electronically senses fluid, it begins to take upfluid (supernatant) from the sample. The tip continues to descend intothe tube while taking up supernatant until it reaches the level in thetube that corresponds to the level of the bar (which corresponds to theinterface or boundary between precipitate and supernatant). At thislevel, the tip stops taking in fluid, and moves vertically upward andout of the sample tube. In this way, a fluid uptake system can removeessentially all of a sample supernatant.

The sample supernatant can be dispensed into a vessel, or dispensed intoanother device or chamber of the automated system.

V. Methods of Using Automated Systems for Enriching Rare Cells of aFluid Sample

The present invention also includes methods of enriching rare cells of afluid sample using an automated system of the present invention. Themethod includes but is not limited to: introducing a sample into anautomated system of the present invention; addition of reagents tosample either before or after the sample is introduced into the system,mixing of sample and reagents; sedimentation of RBCs and removal ofundesirable components; collection of supernatant containing desiredcells; filtering the sample through at least one filtration chamber ofthe automated system; and collecting enriched rare cells from at leastone vessel or at least one outlet of the automated system.

Sample

A sample can be any fluid sample, such as an environmental sample,including air samples, water samples, food samples, and biologicalsamples, including extracts of biological samples. Biological samplescan be blood, a bone marrow sample, an effusion of any type, ascitiesfluid, pelvic wash fluid, pleural fluid, spinal fluid, lymph, serum,mucus, sputum, saliva, urine, vaginal or uterine washes, semen, occularfluid, extracts of nasal, throat or genital swabs, cell suspension fromdigested tissue, or extracts of fecal material. Biological samples canalso be samples of organs or tissues, including tumors, such as fineneedle aspirates or samples from perfusions of organs or tissues.Biological samples can also be samples of cell cultures, including bothprimary cultures and cell lines. The volume of a sample can be verysmall, such as in the microliter range, and may even require dilution,or a sample can be very large, such as up to 10 liters for ascitesfluid. One preferred sample is a urine sample. Another preferred sampleis a blood sample.

A biological sample can be any sample, recently taken from a subject,taken from storage, or removed from a source external to a subject, suchas clothing, upholstery, tools, etc. As an example, a blood sample cantherefore be an extract obtained, for example, by soaking an articlecontaining blood in a buffer or solution. A biological sample can beunprocessed or partially processed, for example, a blood sample that hasbeen dialyzed, had reagents added to it, etc. A biological sample can beof any volume. For example, a blood sample can be less than fivemicroliters, or more than 5 liters, depending on the application.Preferably, however, a biological sample that is processed using themethods of the present invention will be from about 10 microliters toabout 2 liters in volume, more preferably from about one milliliter toabout 250 milliliters in volume, and most preferably between about 5 and50 milliliters in volume.

Introduction of Sample

In some preferred embodiments of the present invention, one or moresamples can be provided in one or more tubes that can be placed in arack of the automated system. The rack can be automatically or manuallyengaged with the automated system for sample manipulations.

Alternatively, a sample can be dispensed into an automated system of thepresent invention by pipetting or injecting the sample through an inletof an automated system, or can be poured, pipetted, or pumped into aconduit or reservoir of the automated system. In most cases, the samplewill be in a tube that provides for optimal separation of sedimentedcells, but it can be in any type of vessel for holding a liquid sample,such as a plate, dish, well, or chamber.

Prior to the dispensing of a sample into a vessel or chamber of theautomated system, solutions or reagents can optionally be added to thesample. Solutions or reagents can optionally be added to a sample beforethe sample is introduced into an automated system of the presentinvention, or after the sample is introduced into an automated system ofthe present invention. If a solution or reagent is added to a sampleafter the sample is introduced into an automated system of the presentinvention, it can optionally be added to the sample while the sample iscontained within a tube, vessel, or reservoir prior to its mixing orincubation step, the settling step, or its introduction into afiltration chamber. Alternatively, a solution or reagent can be added toa sample through one or more conduits, such as tubing, where the mixingof sample with a solution or reagent takes place in conduits. It is alsopossible to add one or more solutions or reagents after the sample isintroduced into a chamber of the present invention (such as, but notlimited to, a filtration chamber), by adding one or more of thesedirectly to the chamber, or through conduits that lead to the chamber.

The sample (and, optionally, any solutions, or reagents) can beintroduced into the automated system by positive or negative pressure,such as by a syringe-type pump. The sample can be added to the automatedsystem all at once, or can be added gradually, so that as a portion ofthe sample is being filtered, additional sample is added. A sample canalso be added in batches, such that a first portion of a sample is addedand filtered through a chamber, and then further batches of a sample areadded and filtered in succession.

Combined Solution for Sedimenting Red Blood Cells and SelectivelyRemoving Undesirable Sample Components of a Blood Sample

In preferred embodiments of the present invention, a solution thatsediments red blood cells can also include one or more additionalspecific binding members that can be used to selectively removeundesirable sample components other than red blood cells from the bloodsample. In this regard, the present invention includes a combinedsedimenting solution for enriching rare cells of a blood sample thatsediments red blood cells and provides reagents for the removal of otherundesirable components of the sample. Thus a combined solution forprocessing a blood sample comprises: dextran; at least one specificbinding member that can induce agglutination of red blood cells; and atleast one additional specific binding member that can specifically bindundesirable components of the sample other than RBCs.

Addition of Sedimenting Solution to Sample

A red blood cell sedimenting solution can be added to a blood sample byany convenient means, such as pipetting, automatic liquiduptake/dispensing devices or systems, pumping through conduits, etc. Theamount of sedimenting solution that is added to a blood sample can vary,and will largely be determined by the concentration of dextran andspecific binding members in the sedimenting solution (as well as othercomponents), so that their concentrations will be optimal when mixedwith the blood sample. Optimally, the volume of a blood sample isassessed, and an appropriate proportional volume of sedimentingsolution, ranging from 0.01 to 100 times the sample volume, preferablyranging from 0.1 times to 10 times the sample volume, and morepreferably from 0.25 to 5 times the sample volume, and even morepreferably from 0.5 times to 2 times the sample volume, is added to theblood sample. (It is also possible to add a blood sample, or a portionthereof, to a red blood cell sedimenting solution. In this case, a knownvolume of sedimenting solution can be provided in a tube or othervessel, and a measured volume of a blood sample can be added to thesedimenting solution.)

Specific Binding Member for Removing Undesirable Components

In addition to the components of a sedimenting solution of the presentinvention, a combined solution of the present invention can comprise atleast one specific binding member that can selectively bind undesirablecomponents of a blood sample (including but not limited to red bloodcells, white blood cells, platelets, serum proteins) and have lessbinding to desirable components. One or more specific binding membersthat can selectively bind undesirable components of a sample can be usedto remove the undesirable components of the sample, increasing therelative proportion of rare cells in the sample, and thus contribute tothe enrichment of rare cells of the sample. By “selectively binds” ismeant that a specific binding member used in the methods of the presentinvention to remove one or more undesirable sample components does notappreciably bind to desirable cells of the sample. By “does notappreciably bind” is meant that not more than 30%, preferably not morethan 10%, and more preferably not more than 1.0% of one or moredesirable cells are bound by the specific binding member used to removeundesirable components from the sample. In many cases, the undesirablecomponents of a blood sample will be white blood cells. In preferredembodiments of the present invention, a combined solution of the presentinvention can be used for sedimenting red blood cells and selectivelyremoving white blood cells from a blood sample.

A specific binding member that can specifically bind white blood cellscan be as nonlimiting examples, an antibody, a ligand for a receptor,transporter, channel or other moiety of the surface of a white bloodcell, or a lectin or other protein that can specifically bind particularcarbohydrate moieties on the surface of a white blood cell (for example,sulfated Lewis-type carbohydrates, glycolipids, proteoglycans orselectin).

Preferably, a specific binding member that selectively binds white bloodcells is an antibody that binds white blood cells but does notappreciably bind fetal nucleated cells, such as, for example, anantibody to CD3, CD11b, CD14, CD17, CD31, CD45, CD50, CD53, CD63, CD69,CD81, CD84, CD102, or CD166. Antibodies can be purchased commerciallyfrom suppliers such as, for example Dako, BD Pharmingen, AntigenixAmerica, Neomarkers, Leinco Technologies, Research & Diagnostic Systems,Serotec, United States Biological, Bender Medsystems Diagnostics,Ancell, Leinco Technologies, Cortex Biochem, CalTag, Biodesign, Biomeda,Accurate Chemicals & Scientific and Chemicon International. Antibodiescan be tested for their ability to bind an efficiently remove whiteblood cells and allow for the enrichment of desirable cells from asample using capture assays well known in the art.

Specific binding members that selectively bind to one or moreundesirable components of the present invention can be used to captureone or more undesirable components, such that one or more desirablecomponents of the fluid sample can be removed from the area or vesselwhere the undesirable components are bound. In this way, the undesirablecomponents can be separated from other components of the sample thatinclude the rare cells to be separated. The capture can be affected byattaching the specific binding members that recognize the undesirablecomponent or components to a solid support, or by binding secondaryspecific binding members that recognize the specific binding membersthat bind the undesirable component or components, to a solid support,such that the undesirable components become attached to the solidsupport. In preferred embodiments of the present invention, specificbinding members that selectively bind undesirable sample componentsprovided in a combined solution of the present invention are coupled toa solid support, such as microparticles, but this is not a requirementof the present invention.

Magnetic beads are preferred solid supports for use in the methods ofthe present invention to which specific binding members that selectivelybind undesirable sample components can be coupled. Magnetic beads areknown in the art, and are available commercially. Methods of couplingmolecules, including proteins such as antibodies, lectins and avidin andits derivatives, to microparticles such as magnetic beads are known inthe art. Preferred magnetic beads of the present invention are from 0.02to 20 microns in diameter, preferably from 0.05 to 10 microns indiameter, and more preferably from 0.05 to 5 microns in diameter, andeven more preferably from 0.05 to 3 microns in diameter and arepreferably provided in a combined solution of the present inventioncoated with a primary specific binding member, such as an antibody thatcan bind a cell that is to be removed from the sample, or a secondaryspecific binding member, such as streptavidin or neutravidin, that canbind primary specific binding members that bind undesirable samplecomponents (such as biotinylated primary specific binding members).

In preferred embodiments of the present invention, the fluid sample is amaternal blood sample, the rare cells whose separation are desirable arefetal cells, and the undesirable components of the sample to be removedfrom the sample are white blood cells and other serum components. Inthese embodiments, a specific binding member that selectively bindswhite blood cells is used to remove the white blood cells from thesample by magnetic capture. Preferably, the specific binding memberprovided is attached to magnetic beads for direct capture, or, isprovided in biotinylated form for indirect capture of white blood cellsby streptavidin-coated magnetic beads.

A combined solution for enriching rare cells of a blood sample of thepresent invention can also include other components, such as, but notlimited to, salts, buffering agents, agents for maintaining a particularosmolality, chelators, proteins, lipids, small molecules,anticoagulants, etc. For example, in some preferred aspects of thepresent invention, a combined solution comprises physiological saltsolutions, such as PBS, PBS lacking calcium and magnesium or Hank'sbalanced salt solution. In some preferred aspects of the presentinvention, EDTA or heparin or ACD are present to prevent red blood cellclotting.

Mixing

The blood sample and red blood cell sedimenting solution are mixed sothat the chemical RBC aggregating agent (such as a polymer, such as, forexample, dextran) and one or more specific binding members of thesedimenting solution, as well as the components of the blood sample aredistributed throughout the sample vessel. Mixing can be achieved meanssuch as electrically powered acoustic mixing, stirring, rocking,inversion, agitation, etc., with methods such as rocking and inversion,that are least likely to disrupt cells, being favored.

Incubation of Blood Sample and Sedimenting Solution

The sample mixed with sedimenting solution is allowed to incubate toallow red blood cells to sediment. Preferably the vessel comprising thesample is stationary during the sedimentation period so that the cellscan settle efficiently. Sedimentation can be performed at anytemperature from about 5° C. to about 37° C. In most cases, it isconvenient to perform the steps of the method from about 15° C. to about27° C. The optimal time for the sedimentation incubation can bedetermined empirically for a given sedimenting solution, while varyingsuch parameters as the concentration of dextran and specific bindingmembers in the solution, the dilution factor of the blood sample afteradding the sedimenting solution, and the temperature of incubation.Preferably, the sedimentation incubation is from five minutes to twentyfour hours in length, more preferably from ten minutes to four hours inlength, and most preferably from about fifteen minutes to about one hourin length. In some preferred aspects of the present invention, theincubation period is about thirty minutes.

Filtering the Sample Through a Chamber of the Automated System

A sample can be filtered in an automated system of the present inventionbefore or after undergoing one or more debulking steps or one or moreseparation steps. These debulking or separation steps can include butare not limited to a RBC sedimentation step or removal by specificbinding members. The sample can be directly transferred to a filtrationchamber (such as by manual or automated dispensing) or can enter afiltration chamber through a conduit. After a sample is added to afiltration chamber, it is filtered to reduce the volume of the sample,and, optionally, to remove undesirable components of a sample. To filterthe sample, fluid flow is directed through the chamber. Fluid flowthrough the chamber is preferably directed by automatic rather thanmanual means, such as by an automatic syringe-type pump. The pump canoperate by exerting positive or negative pressure through conduitsleading to the filtration chamber. The rate of fluid flow through afiltration chamber can be any rate that allows for effective filtering,and for a whole blood sample is preferably between about one and about1000 milliliters per hour, more preferably between about five and about500 milliliters per hour, and most preferably between about ten andabout fifty milliliters per hour. Following the addition of a sample toa filtration chamber, a pump or fluid dispensing system can optionallydirect fluid flow of a buffer or solution into the chamber to washadditional filterable sample components through the chamber.

When the sample is added to the filtration chamber, and fluid flow isdirected through the chamber, pores or slots in the filter or filterscan allow the passage of fluid, soluble components of the samples, andsome non-soluble components of a fluid sample through one or morefilters, but, because of their dimensions, can prevent the passage ofother components of the fluid sample through the one or more filters.

For example, in preferred embodiments a fluid sample can be dispensedinto a filtration chamber that comprises at least one filter thatcomprises a plurality of slots. The chamber can have ports that areoptionally connected to conduits through which a buffer or solution andthe fluid sample or components thereof can flow. When the sample isadded to the chamber, and fluid flow is directed through the chamber,the slots can allow the passage of fluid and, optionally, somecomponents of a fluid sample through the filter, but prevent the passageof other components of the fluid sample through the filter.

In some embodiments of the present invention, an active chip that ispart of the filtration chamber can be used to mix the sample during thefiltration procedure. For example, an active chip can be an acousticchip that comprises one or more acoustic elements. When an electricsignal from a power supply activates the acoustic elements, they providevibrational energy that causes mixing of the components of a sample. Anactive chip that is part of a filtration chamber of the presentinvention can also be a dielectrophoresis chip that comprisesmicroelectrodes on the surface of a filter. When an electric signal froma power supply is transmitted to the electrodes, they provide a negativedielectrophoretic force that can repel components of a sample from thefilter surface. In this embodiment, the electrodes on the surface of thefilter/chip are preferably activated intermittently, when fluid flow ishalted or greatly reduced.

Mixing of a sample during filtration is performed to avoid reductions inthe efficiency of filtration based on aggregation of sample components,and in particular their tendency to collect, in response to fluid flowthrough the chamber, at positions in the chamber where filtering basedon size or shape occurs, such as dams, slots, etc. Mixing can be donecontinuously through the filtration procedure, such as through acontinuous activation of acoustic elements, or can be done in intervals,such as through brief activation of acoustic elements or electrodesduring the filtration procedure. Where dielectrophoresis is used to mixa sample in a filtration chamber, preferably the dielectrophoretic forceis generated in short intervals (for example, from about two seconds toabout 15 minutes, preferably from about two to about 30 seconds inlength) during the filtration procedure; for example, pulses can begiven every five seconds to about every fifteen minutes during thefiltration procedure, or more preferably between about every ten secondsto about every one minute during the filtration procedure. Thedielectrophoretic forces generated serve to move sample components awayfrom features that provide the filtering function, such as, but notlimited to, slots.

During the filtration procedure, filtered sample fluid can be removedfrom the filtration chamber by automated fluid flow through conduitsthat lead to one or more vessels for containing the filtered sample. Inpreferred embodiments, these vessels are waste receptacles. Afterfiltration, fluid flow can optionally be directed in the reversedirection through the filter to suspend retained components that mayhave settled or lodged against the filter.

After the filtration procedure (and optionally, a mixing and incubationwith one or more specific binding members), sample components thatremain in the filtration chamber after the filtration procedure can bedirected out of the chamber through additional ports and conduits thatcan lead to collection tubes or vessels or to other elements of theautomated system for further processing steps, or can be removed fromthe filtration chamber or a collection vessel by pipetting or a fluiduptake means. Ports can have valves or other mechanisms for controllingfluid flow. The opening and closing of ports can be automaticallycontrolled. Thus, ports that can allow the flow of debulked (retained)sample out of a filtration chamber (such as to other chambers orcollection vessels) can be closed during the filtration procedure, andconduits that allow the flow of filtered sample out of a filtrationchamber can optionally be closed after the filtration procedure to allowefficient removal of remaining sample components.

Further Enrichment of Desired Cells

Selective Removal of Undesirable Components of a Sample

Optionally, sample components that remain in the filtration chambereither before, during, or after the filtration procedure can be directedby fluid flow to an element of the automated system in which undesirablecomponents of a sample can be separated from the sample. In someembodiments of the present invention, prior to either adding the sampleto the filtration chamber or removing the debulked sample retained inthe filtration chamber, one or more specific binding members can beadded to the debulked sample and either mixed before the and afterwardsin the filtration chamber, using, for example, one or more active chipsthat engage or are a part of the filtration chamber to provide physicalforces for mixing. Preferably, one or more specific binding member isadded to the debulked sample in the filtration chamber, ports of thechamber are closed, and acoustic elements are activated eithercontinuously or in pulsed, during the incubation of debulked sample andspecific binding members. Preferably, one or more specific bindingmembers are antibodies that are bound to magnetic beads. The specificbinding members can be antibodies that bind desirable sample components,such as fetal nucleated cells, but preferably the specific bindingmembers are antibodies that bind undesirable sample components, such aswhite blood cells while having minimal binding to desirable samplecomponents.

In preferred embodiments of the present invention, sample componentsthat remain in the filtration chamber after the filtration procedure areincubated with magnetic beads, and following incubation, are directed byfluid flow to a separation column. Preferably, a separation column usedin the methods of the present invention is a cylindrical glass, plastic,or polymeric column with a volumetric capacity of between about onemilliliter and ten milliliters, having entry and exit ports at oppositeends of the column. Preferably, a separation column used in the methodsof the present invention comprises or can be positioned alongside atleast one magnet that runs along the length of the column. The magnetcan be a permanent magnet, or can be one or more electromagnetic unitson one or more chips that is activated by a power source.

Sample components that remain in the filtration chamber after thefiltration procedure can be directed by fluid flow to a separationcolumn. Reagents, preferably including a preparation of magnetic beads,can be added to the sample components before or after they are added tothe chamber. Preferably, reagents are added prior to transfer of samplecomponents to a separation chamber. Preferably a preparation of magneticbeads added to the sample comprises at least one specific bindingmember, preferably a specific binding member that can directly bind atleast one undesirable component of the sample. However, it is alsopossible to add a preparation of magnetic beads that comprise at leastone specific binding member that can indirectly bind at least oneundesirable component of the sample. In this case, it is necessary toalso add a primary specific binding partner that can directly bindundesirable components to the sample. A primary specific binding partneris preferably added to the sample before the preparation of magneticbeads comprising a secondary specific binding partner is added to thesample, but this is not a requirement of the present invention. Beadpreparations and primary specific binding partners can be added to asample before or after the addition of the sample to a separationcolumn, separately or together.

In embodiments where magnetic beads comprise primary specific bindingmembers, the sample and magnetic bead preparation are preferablyincubated together for between about five and about sixty minutes beforemagnetic separation. In embodiments where a separation column comprisesor is adjacent to one or more permanent magnets, the incubation canoccur prior to the addition of the sample to the separation column, inconduits, chambers, or vessels of the automated system. In embodimentswhere a separation column comprises or is adjacent to one or morecurrent-activated electromagnetic elements, the incubation can occur ina separation column, prior to activating the one or more electromagneticelements. Preferably, however, incubation of a sample with magneticbeads comprising specific binding members occurs in a filtration chamberfollowing filtration of the sample, and after conduits leading into andout of the filtration chamber have been closed.

Where magnetic beads comprising secondary specific binding members areemployed, optionally more than one incubation can be performed (forexample, a first incubation of sample with a primary specific bindingmember, and a second incubation of sample with beads comprising asecondary specific binding member). Separation of undesirable componentsof a sample can be accomplished by magnetic forces that cause theelectromagnetic beads that directly or indirectly bind the undesirablecomponents. This can occur when the sample and magnetic beads are addedto the column, or, in embodiments where one or more electromagneticunits are employed, by activating the electromagnetic units with a powersupply. Noncaptured sample components can be removed from the separationcolumn by fluid flow. Preferably, noncaptured sample components exit thecolumn through a portal that engages a conduit.

Separation of Desirable Components

After filtering, a sample can optionally be directed by fluid flow to aseparation chamber for the separation of rare cells.

In preferred aspects in which undesirable components of a debulkedsample have been removed in a separation column, the debulked sample ispreferably but optionally transferred to a second filtration chamberprior to being transferred to a separation chamber for separation rarecells of the sample. A second filtration chamber allows for furtherreduction of the volume of a sample, and also optionally allows for theaddition of specific binding members that can be used in the separationof rare cells and mixing of one or more specific binding members with asample. Transfer of a sample from a separation column to a separationchamber is by fluid flow through conduits that lead from a separationcolumn to a second filtration chamber. A second filtration chamberpreferably comprises at least one filter that comprises slots, and fluidflow through the chamber at a rate of between about one and about 500milliliters per hour, more preferably between about two and about 100milliliters per hour, and most preferably between about five and aboutfifty milliliters per hour drives the filtration of sample. In this way,the volume of a debulked sample from which undesirable components havebeen selectively removed can be further reduced. A second filtrationchamber can comprise or engage one or more active chips. Active chips,such as acoustic chips or dielectrophoresis chips, can be used formixing of the sample prior to, during, or after the filtrationprocedure.

A second filtration chamber can also optionally be used for the additionof one or more reagents that can be used for the separation of rarecells to a sample. After filtration of the sample, conduits that carrysample or sample components out of the chamber can be closed, and one ormore conduits leading into the chamber can be used for the addition ofone or more reagents, buffers, or solutions, such as, but not limitedto, specific binding members that can bind rare cells. The one or morereagents, buffers, or solutions can be mixed in the closed-offseparation chamber, for example, by activation of one or more acousticelements or a plurality of electrodes on one or more active chips thatcan produce physical forces that can move components of the sample andthus provide a mixing function. In preferred aspects of the presentinvention, magnetic beads that are coated with at least one antibodythat recognizes a rare cell are added to the sample in the filtrationchamber. The magnetic beads are added via a conduit, and are mixed withthe sample by activation of one or more active chips that are integralto or engage a second filtration chamber. The incubation of specificbinding members with a sample can be from about five minutes to abouttwo hours, preferably from about eight to about thirty minutes, induration, and mixing can occur periodically or continuously throughoutthe incubation.

It is within the scope of the present invention to have a secondfiltration chamber that is not used for the addition and mixing of oneor more reagents, solutions, or buffers with a sample. It is also withinthe scope of the present invention to have a chamber that precedes aseparation chamber for the separation of rare cells that can be used forthe addition and mixing of one or more reagents, solutions, or bufferswith a sample, but that does not perform a filtering function. It isalso within the scope of the present invention to have a sampletransferred from a separation column to a separation chamber, without anintervening filtration or mixing chamber. In aspects where the methodsare directed toward the separation of rare cells from a blood sample,however, the use of a second filtration chamber that is also used forthe addition and mixing of one or more reagents with a sample ispreferred.

Sample is transferred to a separation chamber by fluid flow. Preferably,a separation chamber for the separation of rare cells comprises orengages at least one active chip that can perform a separation. Suchchips comprise functional elements that can, at least in part, generatephysical forces that can be used to move or manipulate sample componentsfrom one area of a chamber to another area of a chamber. Preferredfunctional elements of a chip for manipulating sample components areelectrodes and electromagnetic units. The forces used to translocatesample components on an active chip of the present invention can bedielectrophoretic forces, electromagnetic forces, traveling wavedielectrophoretic forces, or traveling wave electromagnetic forces. Anactive chip used for separating rare cells is preferably part of achamber. The chamber can be of any suitable material and of any size anddimensions, but preferably a chamber that comprises an active chip usedfor separating rare cells from a sample (a “separation chamber”) has avolumetric capacity of from about one microliter to ten milliliters,more preferably from about ten microliters to about one milliliter.

In some embodiments of the present inventions, the active chip is adielectrophoresis or travelling wave dielectrophoresis chip thatcomprises electrodes. Such chips and their uses are described in U.S.application Ser. No. 09/973,629, entitled “An Integrated Biochip Systemfor Sample Preparation and Analysis”, filed Oct. 9, 2001; U.S.application Ser. No. 09/686,737, filed Oct. 10, 2000 entitled“Compositions and Methods for Separation of Moieties on Chips”, U.S.application Ser. No. 09/636,104, filed Aug. 10, 2000, entitled “Methodsfor Manipulating Moieties in Microfluidic Systems”; and U.S. applicationSer. No. 09/679,024 having attorney docket number 471842000400, entitled“Apparatuses Containing Multiple Active Force Generating Elements andUses Thereof” filed Oct. 4, 2000; all incorporated by reference. Rarecells can be separated from a sample of the present invention by, forexample, their selective retention on a dielectrophoresis chip, andfluid flow can remove non-retained components of the sample.

In other preferred embodiments of the present invention, the active chipis an electromagnetic chip that comprises electromagnetic units, suchas, for example, the electromagnetic chips described in U.S. Pat. No.6,355,491 entitled “Individually Addressable Micro-Electromagnetic UnitArray Chips” issued Mar. 12, 2002 to Zhou et al., U.S. application Ser.No. 09/955,343 having attorney docket number ART-00104.P.2, filed Sep.18, 2001, entitled “Individually Addressable Micro-Electromagnetic UnitArray Chips”, and U.S. application Ser. No. 09/685,410 having attorneydocket number ART-00104.P.1.1, filed Oct. 10, 2000, entitled“Individually Addressable Micro-Electromagnetic Unit Array Chips inHorizontal Configurations”. Electromagnetic chips can be used forseparation by magnetophoresis or traveling wave electromagnetophoresis.In preferred embodiments, rare cells can be incubated, before or afteraddition to a chamber that comprises an electromagnetic chip, withmagnetic beads comprising specific binding members that can directly orindirectly bind the rare cells. Preferably, in embodiments where rarecells are captured on an electromagnetic chip, the sample is mixed withthe magnetic beads comprising a specific binding member in a mixingchamber. Preferably, a mixing chamber comprises an acoustic chip for themixing of the sample and beads. The cells can be directed throughconduits from the mixing chamber to the separating chamber. The rarecells can be separated from the fluid sample by magnetic capture on thesurface of the active chip of the separation chamber, and other samplecomponents can be washed away by fluid flow.

The methods of the present invention also include embodiments in whichan active chip used for separation of rare cells is a multiple-forcechip. For example, a multiple-force chip used for the separation of rarecells can comprise both electrodes and electromagnetic units. This canprovide for the separation of more than one type of sample component.For example, magnetic capture can be used to isolated rare cells, whilenegative dielectrophoresis is used to remove undesirable cells from thechamber that comprises the multiple-force chip.

After the removal of undesirable sample components from the separationchamber, either through active physical forces such as negativedielectrophoresis or by fluid flow, the captured rare cells can berecovered by removing the physical force that causes them to adhere tothe chip surface, and collecting the cells in a vessel using fluid flow.

EXAMPLES Example 1 Fabrication of a Filter for Removing Red Blood Cellsfrom a Blood Sample

A silicon chip of dimensions (1.8 cm by 1.8 cm×500 micron) was used tofabricate a filtration area of 1 cm by 1 cm by 50 micron with slotshaving dimensions from about 0.1 micron to about 1000 microns,preferably from about 20 to 200 microns preferably from about 1 to 10microns, more preferably 2.5 to 5 microns. The slots were verticallystraight with a maximum tapered angle of less than 2%, preferably lessthan about 0.5% with an offset distance between neighboring columns ofthe filter slots were 1 to 500 microns, preferably from 5 to 30 microns.

Manufacturing included providing a silicon chip having the abovereferenced dimensions and coating the top and bottom of the silicon chipwith a dielectric layer. A cavity along the bottom portion of the chipwas then created. The cavity was formed by removing an appropriatecavity pattern from the dielectric layer then etching the silicon chipgenerally following the pattern until desired thickness is reached. Thechip was reoxidized to coat the contoured region. A filter pattern wasthen removed from the dielectric layer coating the top of the siliconchip in substantial alignment (above) with the cavity. The silicon chipwas etched (e.g. via deep RIE or ICP processes) at the above referencedangles starting at the pattern created along the top of the chip untilthe silicon layer has been etched through. The dielectric layer from thetop and bottom were then removed. By removing the dielectric layerwithin the cavity, throughbores, referred to as slots, were created. Itis also possible to create these slots using laser cuts to bore thoughmaterials, including but not limited to silica or polymers such asplastic.

Example 2 Chemical Treatment of a Microfabricated Filter

A filter chip made as described in Example 1 was placed on a ceramicheating plate in an oven and heated at 800 degrees Celsius for 2 hoursin oxygen containing gas (e.g. air). The heating source was then turnedoff the chips are slowly cooled overnight. This results in a thermallygrown layer on the surface of the chip.

A nitride layer could also be deposited onto the filter surface. Anoxide layer is put on the surface of the chip by low-pressure chemicalvapor deposition (LPCVD) in a reactor at temperatures up to ˜900° C. Thedeposited film is a product of a chemical reaction between the sourcegases supplied to the reactor. The process is typically performed onboth sides of the substrate at the same time to form a layer of Si3N4.

Example 3 Polyvinylpyrrolidone (PVP) and Polyvinyl Alcohol (PVA) FilterCoatings

Filter chips made by the method of Example 1 were coated with either PVPor PVA. For coating the chips with either PVP or PVA, the chips werepre-treated as follows: The filter chips were rinsed with deionizedwater and then immersed in 6N nitric acid. The chips were placed on ashaker for 30 minutes at 50 degrees Celsius. After acid treatment, thechips were rinsed in deionized water.

For PVP coating, chips were immersed in 0.25% polyvinylpyrrolidone(K-30) at room temperature until the chips were ready for use. Chipswere then rinsed with deionized water and dried by pressurized air.

For PVA coating, after acid treatment and rinsing in water, the chipswere stored in water prior to coating. To make the 0.25% PVA (Mn35,000-50,000) solution, dissolve the PVA in water under slow heating to80 degrees Celsius and gentle stirring. To coat, the chips were immersedin a hot PVA solution and heated for 1-2 hours. The chips were thenrinsed in deionized water and dried by pressurized air.

Example 4 Bovine Serum Albumin (BSA) Filter Coating

For coating filter chips with BSA, the chips were pre-treated asfollows: The filter chips were rinsed with deionized water and thenimmersed in 95% ethanol for 10 seconds at room temperature and then wererinsed again in deionized water.

The chips were then immersed in 2.% BSA in PBS for 2 minutes at roomtemperature. Chips were then rinsed with deionized water and dried bypressurized air.

Example 5 PEG Filter Coating

To conjugate PEG to the chip surfaces, filter chips were immersed in asolution of DBE-814 (a PEG solution containing polysiloxane from Gelest,Morrisville, Pa.) in 5% methylene chloride. The immersed chips wereheated at 70 degrees Celsius for 3 hours under vacuum. After theincubation, the PEG-coated chips were rinsed in deionized water anddried by pressurized air.

Example 6 Procedure for Enriching Fetal Cells from Maternal Blood

We developed a two-step procedure for enrichment of fetal cells frommaternal blood.

Step One: Blood Debulking and WBC Removal.

(1) A Combined Reagent:

The combined reagent has two components:

a) RBC aggregation solution

-   -   2% Dextran (110,000 MW)    -   0.05 ugs/ml of IgM antibody to glycophorin A    -   5 mM EDTA    -   1×PBS without calcium and magnesium.        The RBC aggregation solution works with heparin or ACD instead        of EDTA. The RBC aggregation solution also works with a base        solution including but not limited to lxHanks balanced saline        solution with heparin, ACD, or EDTA. The concentration of IgM        antibody to glycophorin A can vary from 0.05 to 0.15 (range of        0.01 to 10) ug/ml.

b) WBC depletion solution

-   -   Magnetic beads (1.0 micron magnetic beads prepared by AVIVA),        precoated with antibody (5-60 ugs per 10⁹ beads)        The combined reagent has the RBC aggregation solution with 30        (range of 5-60) antibody precoated magnetic beads per WBC and        can include but not limited to magnetic beads precoated with        antibodies or other reagents to specifically bind other        components of blood.

(2) Use of the Combined Reagent:

The combined reagent was added to an equal volume of washed peripheralblood and incubated with rotation for 0.5 hr (range of 0.1-2 hours). Thetube was settled for 0.5 hr (range of 0.1 to 2 hrs) upright against amagnet (Dynal, MPC-1). We have also tested a magnet on the bottom of thetube as well.

The solution from the top portion of the tube that did not includeaggregated or magnetically captured cells (on the side of the tube or atthe bottom portion of the tube) was aspirated off and transferred to anew tube.

Step Two: Further Enrichment of Nucleated Cells and Removal of RBCs.

The aspirated solution can be then further processed to enrich fornucleated fetal cells and remove RBCs by either a magnetic separationstep (antibody to CD71 with MACS microbeads) or a microfiltration step.[Table 10] shows the results of using the above described first stepfollowed by CD71 antibody capture of fetal cells. [Table 10] (below)shows the results of using a combined solution (step one describedabove) followed by microfiltration.

TABLE 10 nRBC recovery comparing CD71 vs filter chip. ExperimentComparing CD71 and Silicon Membrane for capture fetal nRBC SamplesMB24479,11 wk gestation presurgery, MB24481, 12 wk gestation drawn andarrived on Jun. 25, 2002. Date Jun. 25, 2002 Prepacyte Sample 1 Sample 2Sample 3 Sample 4 Procedure and Results Number of times 3 3 3 3 washedSample name MB24479 MB24479 MB24481 MB24481 Sex of fetus Male MaleFemale Female Number of WBC/ml 9.60E+06 9.60E+06 9.40E+06 9.40E+06 Startsamples in mls 10  10  10  10  PBS with EDTA 8 8 8 8 10% Dextrans 110 in2 2 2 2 PBS-EDTA lgM GpA 0.5 μg 0.5 μg 0.5 μg 0.5 μg Bead ManufactorAviva Beads/WBC 30  30  30  30  Bead Lot Apr. 24, 2002 NAV beads Apr.24, 2002 NAV beads Apr. 24, 2002 NAV beads Apr. 24, 2002 NAV beads with30 μg CD50 with 30 μg CD50 with 30 μg CD50 with 30 μg CD50 biotin/1 ×10{circumflex over ( )}9 beads biotin/1 × 10{circumflex over ( )}9 beadsbiotin/1 × 10{circumflex over ( )}9 beads biotin/1 × 10{circumflex over( )}9 beads Rock 30 min at RT in one 50 ml conical and 5 ml tube Stand30 min at RT with a magnet Standing Magnet Sep Dynal Magnet wash 2X @1200 rpm 2X @ 1200 rpm CD71 enrichment or Silicon Membrane volume 1 mlTaiwan silicon 1 ml Taiwan silicon CD71 0.1 μg membrane 11_2 at 0.1 μgmembrane 11_2 at Time/Tem 15 min at RT flow rate of 20 mls 15 min at RTflow rate of 20 mls Step 2: add beads per hour with a per hour with avolume 1 ml magnetic trap 1 ml magnetic trap MAC SAV Beads 100 μl 100 μlTime/Tem 15 min at RT 15 min at RT Total Cells Remaining 7.00E+056.60E+05 6.80E+05 8.80E+05 slides proposed to 7 6 7 8 make Actual sidesmade 7 6 7 8 nRBCs counted 4, 3, 0 3, 3 0, 4, 2 4, 2 estimated totalnRBC 16  18  14  24 

Example 7 Process Flow Chart for Enriching Nucleated Fetal Cells fromMaternal Blood

FIG. 13 shows a process flow chart for enriching fetal nucleated cellsfrom maternal blood samples. The whole process comprises the flowingsteps:

-   -   (1) The blood sample may be transferred to a centrifuge tube.    -   (2) The sample does not have to be but can be washed before        addition to the automated unit.    -   (3) The process starts with a volume of blood sample 10 mis        (range of 3-40 ml) in a tube(s).    -   (4) Fluidic level sensing step is used to determine the exact        volume of the blood sample in the tube to be processed.    -   (5) Add a volume of the combined reagent (for example, an equal        volume of the reagent described in Example 6) to the blood        sample in the tube.    -   (6) Rotate/shake/tumble/mix the solution for a period of time        0.5 hrs (range of 0.1-2 hrs).    -   (7) Let the solutions in the tube settle upright for 30 minutes        (range of 0.1 to 2 hrs) so that the aggregated RBCs can settle        to the bottom of the tube. Simultaneously during this period, a        magnetic field is applied to collect and attract magnetic beads        (which may or may not have bound blood components) to a side of        tube.    -   (8) Another fluidic level sensing step is applied to determine        what the volume of the “un-aggregated” cell suspension is        present in the tube.    -   (9) Aspirate appropriate volume of the fluid from the tube into        the fetal cell filtration chamber (or fetal cell cassette        process).    -   (10) Filter the sample for 0.2-2 hr in the fetal cell filtration        chamber/cassette (Further details of the filtration process are        included in [Example 9], below.)    -   (11) Extract solution from the top chamber of the filtration        cassette and dispense into storage test tube.

Example 8 Process Flow Chart for Silicon Membrane Filtration Process

FIG. 14 provides a schematic diagram showing the microfiltrationprocess. The simplified process steps include the following:

-   -   (1) Close valves B&D, open valves A&C.    -   (2) Test sample (coming from the first step of the procedure in        [Example 10]) is loaded into the 45 mL loading reservoir.    -   (3) Operate waste pump for 1 h so that the sample loaded in the        storage reservoir is filtered Through the microfabricated        filter.    -   (4) Apply 1-10 mL wash solution to the Loading Reservoir.    -   (5) Close valve A, open valve B.    -   (6) Wash the bottom subchamber with 1-5 mL.    -   (7) Close valve C and open valve D.    -   (8) Rotate the Cassette and filtration chamber 180 degrees.    -   (9) Flush the filter from valve B.    -   (10) Collect volume from valve D.

Example 9 Use of an Automated System to Isolate Fetal Cells fromMaternal Blood

Ten milliliters blood samples of pregnant women (from six to thirtyweeks gestation) are washed by diluting the samples with PBE andcentrifuged at 470×g for 6 minutes (range of 50-900×g for 3-20 minutes).The supernatants are aspirated off, and PBE is added to the pellets andmixed. The samples are again centrifuged and the supernatants aspiratedoff. The final pellets are resuspended to the original volume with PBE.Ten milliliters of Combined Reagent (PBS lacking calcium and magnesiumcontaining: 5 millimolar EDTA, 2% dextran (molecular weight from 70 to200 kilodaltons), 0.05 micrograms (range of 0.01 to ugs) per milliliterof IgM antibodies to glycophorin A, and approximately 1-10×10⁹ precoatedmagnetic beads are manually added to the sample tubes.

The Rare Cell Isolation Automated System has control circuits forautomated processing steps, and plugs into a 110 volt outlet. The tubescontaining the samples are placed in a rack of a Rare Cell IsolationAutomated System. The tubes are automatically rotated in the AutomatedSystem rack for 30 minutes (range between 5 and 120 minutes). The tubesare then allowed to stand upright while a second rack that has a magnetfield, which is automatically positioned next to the tube rack. It isalso possible to have other types of magnetic fields including but notlimited to electromagnetic fields. The tubes are held in the uprightposition for 30 minutes (range of 5-120 minutes) so that the aggregatedRBCs can settle to the bottom of the tube and WBC-magnetic beadaggregates are attracted to the side of each tube that is adjacent tothe magnet. After the cells are allowed to settle, the supernatantvolume is determined by the automated system using a lighttransmission-light sensor transparency measuring device.

The transparency measuring device consists of bars that each have acollated light source (the number of bars corresponds to the number oftubes) that can be focused on a sample tube, and a light detector thatis positioned on the opposite side of the tube. The light source uses alaser beam that emits light in the infrared range (780 nanometers) andhas an intensity greater than 3 milli-watts. The light from the sourceis focused through the sample tube, and at the other side of the sampletube the light detector having an intensity measurement device recordsthe amount of light that has passed through the sample (the laser outputmeasurement). The bars having the low wattage laser sources and lightdetectors move upward from a level at the bottom of the tubes. As eachlaser makes initial contact with the aggregated cells in thecorresponding tube, the laser output measurement is zeroed. When themeasured intensity for a given tube begins to rise above a thresholdvalve the vertical movement of the bar stops. The bar then moves to findthe exact vertical point at which the transmitted light equals thethreshold value. In this way the vertical point position of theaggregated cell/cell supernatant interface is determined. Once thislevel has been determined, the fluid handling unit moves to a presetlocation and uses a capacitive sensing routine to find the level of thebar (corresponding to the level of the interface). Using this data, thefluid handling accurately removes the supernatant from the fluidcontainer. The supernatant is automatically dispensed directly into theloading reservoir of the filtration unit.

The following description of the automated separation process performedby the Rare Cell Isolation Automated System uses a filtration unit(filtration chamber, loading reservoir, and associated ports and valves)as depicted in FIG. 23. In this design, the filtration chamber canrotate 180 degrees or more within the filtration unit.

The filtration chamber comprises an antechamber (604) and apostfiltration subchamber (605) separated by a single filter (603). Themicrofabricated filter measuring 1.8 cm by 1.8 cm and having afiltration area of approximately 1 cm by 1 cm. The filter hasapproximately 94,000 slots arranged in a parallel configuration as shownin FIG. 2 with the slots having a taper of one to two degrees anddimensions of 3 microns×100 microns, within a 10% variation in eachdimension. The filter slots can have dimensions of 1-10 microns by10-500 microns with a vertical taper of 0.2 to 10 degrees depending onthe target. The thickness of the filter is 50 microns (range of 10-200microns). The filter is positioned in a two piece filtration chamberwith the top half (antechamber) being an approximately rectangularfiltration antechamber that tapers upward with a volume of approximately0.5 milliliters. The bottom post-filtration subchamber is alsoapproximately circular and tapers toward the bottom, also having avolume of approximately 0.5 milliliters. The filter covers essentiallythe entire bottom area of the (top) antechamber and essentially theentire top area of the (bottom) postfiltration subchamber.

In addition to the filtration chamber, the filtration unit comprises a“frame” having a loading reservoir (610), a valve controlling the flowof sample form the loading reservoir into the filtration chamber (“valveA”, 606), and separate ports for the outflow of waste or filtered sample(the waste port, 634) and for the collection of enriched rare cells (thecollection port, 635). The post-filtration subchamber (605) comprises aside port (632) that can be used for the addition of buffer, and anoutlet that can engage the waste port during filtration for the outflowof waste (or filtered sample). The antechamber (604) comprises an inletthat during filtration can engage the sample loading valve (valve A,606) and during collection of enriched cells, can engage the collectionport (635). During operation of an automated system, the filtrationchamber (comprising the antechamber (604), post-filtration subchamber(605), and side port (632)) resides in the frame of the filtration unit.

During filtration, valve A is open, and the outlet of thepost-filtration subchamber is aligned with the waste port, allowing aflow path for filtering sample from the loading reservoir through thefiltration chamber and to the waste. A syringe pump draws fluid throughthe chamber at a flow rate of from about 10 to 500 milliliters per hour,depending upon the process step.

Prior to dispensing the appropriate volume of supernatant from each tubeinto the loading reservoir of the filtration unit, the side port (632)and waste port (634) of the filtration unit are closed, and valve A(606) is opened (see FIG. 23). (When the filtration unit is in theloading/filtering position, the filtration chamber does not engage thecollection port (635)). With the side port of the filtration unit open,the unit is filled with PBE from the side port until the buffer reachesthe bottom of the sample reservoir. The side port is then closed, andthe blood sample supernatant is loaded into the loading reservoir.

Although the Rare Cell Isolation Automated System can separate severalsamples simultaneously, for clarity, the description of the separationprocess that follows will describe the filtration of a single sample. Tofilter a sample, the waste port (634) of a filtration unit is opened,and, using a syringe pump connected through tubing to the waste port,sample supernatant is drawn into and through the filtration chamber. Assample goes through the chamber, the larger cells stay in the topchamber (antechamber) and the smaller cells go through the filter intothe lower chamber (post-filtration subchamber) and then through thewaste port to the waste. Filtering is performed at a rate ofapproximately 10-100 milliliters per hour.

After a sample has gone through a filtration chamber (typically afterfrom one half to two hours of filtering), three to five milliliters ofPBE are added to the loading reservoir (with valve A remaining open) andpulled through the filtration chamber using the syringe pump connectedto the waste port to wash the antechamber and make sure virtually allsmall cells are washed through.

Valve A (606) is then closed and the side port (632) is opened. Five toten milliliters of buffer are added from the side port (632) using asyringe pump connected to tubing that is attached to the waste port(634) to wash the bottom post-filtration subchamber. After residualcells have been washed from the post-filtration subchamber (605), thebottom (post-filtration) subchamber is further cleaned by pushing airthrough the side port (632).

The filter cartridge is then rotated approximately 180 degrees withinthe filtration unit, so that the antechamber (604) is below thepost-filtration subchamber (605). When the chamber rotates intocollection position, the outlet of the post-filtration subchamberdisengages from the waste port and, as the post-filtration subchamberbecomes positioned above the antechamber, the “outlet” becomespositioned at the top of the inverted filtration chamber, but does notengage any openings in the filtration unit, and thus is blocked. As thishappens, the antechamber rotates to the bottom of the invertedfiltration unit, so that the antechamber inlet disengages from valve A,and instead engages the collection port at the bottom of the filtrationunit. During this rotation from the filtering position to the collectionposition, the side port does not change position. It is aligned with theaxis of rotation of the filtration chamber, and remains part of, and afunctional port of, the post-filtration subchamber. As a result of thisrotation, the filtration chamber is in the collection position. Thus, inthe collection position, the post-filtration subchamber, having a sideport but now closed off at its outlet, is above the antechamber. Theantechamber “inlet” is aligned with and open to the collection port.

Approximately two milliliters of buffer is pumped into the filtrationchamber through the side port to collect the cells left in theantechamber. The cells are collected into a vial that attaches to thefiltration unit at the site of the sample collection port, or via tubingthat leads from the sample collection port and dispenses the sample intoa collection tube. Approximately 2 milliliters of additional PBE, andapproximately 2 to 5 milliliters of air, is pumped through the side portto clean residual cells off of the filter and into the collection vial.

The enriched rare cells can be analyzed microscopically or using any ofa number of assays, or can be stored or put into culture.

Example 10 Improved Magnet Configurations for Magnetic Particle Capture

To improve the efficiency of separating components such as cells fromliquid samples using capture of magnetic particles to one portion of atube or other container, several magnet configurations were tested.

Magnets of dimensions 9/16×1.25×⅛″, (Forcefield (Fort Collins, Co) NdFeBblock, item #27, Nickel Plate, Br max 12,100 Gauss, Bh max 35 MGOe) wereused to test the magnetic field strength. In these experiments, thestrongest field could be used to capture magnetic beads that were coatedwith antibodies that specifically bound white blood cells, and improvethe removal of white blood cells from a blood sample compared tocommercially available magnetic cell separation unit MPC-1 (Dynal,(Brown Deer, Wis.).

Magnets were attached in several configurations and orientations to apolypropylene stand designed to hold a 50 milliliter tube, as depictedschematically in Figure [X]. The magnetic field in the right, center,and left of the tube was measured by Gauss meter (JobMaster Magnets(Randallstown, Md.) Model GM1 using probe model PT-70, Cal #373).

Example 11 Depletion of Platelets from a Blood Sample with Antibody toCD31

To remove platelets from a peripheral blood sample, 1.5 to 4×10⁹neutravidin coated magnetic beads (AVIVA Biosciences Corp) were coatedwith 75 micrograms of biotinylated CD31 antibody (AVIVA SystemsBiology). The beads were washed and resuspended in PBE (PBS containing0.5% BSA and 5 mM EDTA) to a concentration of 4×10⁹ beads permilliliter.

For each 10 milliliter of washed blood sample, 1.5 to 4×10⁹ ofCD31-coated magnetic beads were used. The beads were added to 10milliliters of washed blood sample in a 50 milliliter polypropylenetube. The tube was rotated for 30 minutes (range of 5 to 120 minutes)and then placed in a stand having one or more magnets positioned alongthe outside of the tube. After thirty minutes (range of 5 to 120minutes), the supernatant was removed.

The samples were filtrated using a micro-fabricated filter and the cellswere recovered. The cells were resuspended to 10⁶ cells per milliliterand spun onto a slide. The slides were stained using a BenzidineWright-Giemsa staining protocol and the slide was visually analyzed.Photographs of slides are provided as FIG. 25. FIG. 25A being a controland FIG. 25B being a sample treated with CD31 antibody.

Example 12 Selection of Gestational Age for Blood Sample Collection forFetal Cell Isolation from Maternal Blood Samples

Blood samples were drawn from pregnant women at from about 11 to 16weeks of gestation age. Samples were centrifuged at 1500 rpm (470×g) for6 minutes and the supernatant aspirated away from the cell pellet, thecell pellet was resuspended in PBE and centrifuged for further washing.The second centrifugation step was done at two speeds, either 1500 rpm(470×g) for 10 minutes or 1000 rpm (209×g) for 2 minutes. The effect onfetal cell recovery is described in the following table:

Fetal cells recovered Gest 2^(nd) Centrifugation Sample AnticoagulantAge 1500 × 10 1000 × 2 W2 Final 7615 Heparin 15 2 1 0 2 7623 Heparin 132 3 0 2 7624 Heparin 12 1 3 2 3 7629 Heparin 14 0 1 0 0 7558 Heparin 143 7 1 3 7655 ACD 13 6 2 0 6 7674 ACD 16 4 0 3 4 7716 ACD 11 1 5 6 5Total enriched fetal cells 19 22 25The column labeled “W2” is the amount of fetal cells recovered from thesupernatant after the second centrifugation step. The columns labeled“1500×10” and “1000×2” were the two centrifugation speeds chosen for thesecond centrifugation step. The column labeled “Final” was the amount offetal cells enriched using the following parameter: if a samples had agestation age of less than 13 weeks then the amount of fetal cellsrecovered was chosen from the column labeled 1000 rpm (209×g) for 2 min;and the samples had a gestation age of greater than or equal to 13weeks, then the amount of fetal cells recovered was chosen from thecolumn labeled 1500 rpm (470×g) for 10 min.

Example 13 Centrifugation Conditions for Fetal Cell Isolation fromMaternal Blood Samples

Blood samples were drawn from pregnant women at about 11 to about 20weeks of gestation. Samples of about 10 milliliters were centrifuged at1500 rpm (470×g) for 6 minutes to wash the cells. The pellet wasresuspended in approximately 45 milliliters of PBE and then centrifugedonce more as a second wash at two speeds either 1500 rpm (470×g) for 10minutes or 1000 rpm (209×g) for 2 minutes.

Enrichment of fetal nucleated RBCs from maternal blood was performed asfollows:

Step One: Blood Debulking and WBC Removal.

(1) a Combined Reagent:

The combined reagent has two components:

c) RBC aggregation solution

-   -   2% Dextran (110,000 MW)    -   0.05 ugs/ml of IgM antibody to glycophorin A    -   5 mM EDTA    -   1×PBS without calcium and magnesium.        The RBC aggregation solution works with other saline based        solutions including but not limited to 1× Hanks balanced saline        solution. The anticoagulant can include but not limited to        heparin, ACD or EDTA The concentration of antibody to        glycophorin A can have a range of 0.01 to 10 ugs/ml.

d) WBC depletion solution

-   -   Magnetic beads (1.0 micron magnetic beads prepared by AVIVA),        precoated with antibody (5-60 ugs per 10⁹ beads)        The combined reagent has the RBC aggregation solution with 15-60        precoated magnetic beads per WBC.

(2) Use of the Combined Reagent:

The combined reagent was added to an equal volume of washed peripheralblood and incubated with rotation for 30 minutes (range of 0.1-2 hour).The tube was settled for 0.5 hr (range of 0.1 to 2 hours) uprightagainst a magnet (Dynal, MPC-1). We have also tested a magnet on thebottom of the tube as well.

The solution from the top portion of the tube that did not includeaggregated cells (on the side of the tube or at the bottom portion ofthe tube) was aspirated off and transferred to the next step.

Step Two: Further Enrichment of Fetal Cells and Removal of RBCs.

The aspirated solution can be then further processed to enrich fornucleated fetal cells and remove RBCs by either a magnetic separationstep (e.g. antibody to CD71 with MACS microbeads) or a microfiltrationstep.

The column labeled “W2” is the amount of fetal cells recovered from thesupernatant after the second centrifugation step. The columns labeled“1500×10” and “1000×2” were the two centrifugation speeds chosen for thesecond centrifugation step. The column labeled “Final” was the amount offetal cells enriched using the following parameter: if a samples had agestation age of less than 13 weeks then the amount of fetal cellsrecovered was chosen from the column labeled 1000 rpm (209×g) for 2 min;and the samples had a gestation age of greater than or equal to 13weeks, then the amount of fetal cells recovered was chosen from thecolumn labeled 1500 rpm (470×g) for 10 min.

Results:

Gest Sample Anticoagulant Age 1500 × 10 1000 × 2 W2 Final 7615 Heparin15 2 1 0 2 7623 Heparin 13 2 3 0 2 7624 Heparin 12 1 3 2 3 7629 Heparin14 0 1 0 0 7558 Heparin 14 3 7 1 3 7655 ACD 13 6 2 0 6 7674 ACD 16 4 0 34 7716 ACD 11 1 5 6 5 19 22 25

Example 14 Fetal Cells Isolated from Maternal Blood

During enrichment of rare fetal cells using methods disclosed herein,the sample and various sample fractions were tested for the presence andabundance of nucleated fetal cells. This is presented schematically inFIG. 24. The figure shows a fetal cell enrichment procedure that beginswith a maternal blood sample (upper left) and ends in a high-qualitypreparation of enriched fetal cells.

The steps of the enrichment procedure, going in sequential order andfrom upper left to lower right in the figure, are: 1) washing the bloodsample (0-2 centrifugations); 2) selectively sedimenting red blood cellsand selectively removing white blood cells with a Combined Reagent (PBSlacking calcium and magnesium containing: 5 millimolar EDTA, 2% dextran(molecular weight from 70 to 200 kilodaltons), 0.05 micrograms (range of0.01 to 10) per milliliter of IgM antibodies to glycophorin A, andapproximately 1-5×10⁹ magnetic beads coated with a CD50 antibody); and3) filtering the supernatant of step 2) through a microfabricatedfilter, such as the microfabricated filters described in Examples 15 and16.

In FIG. 24, “AVIPrep” refers a red blood cell sedimenting solution, forexample, PBS lacking calcium and magnesium containing: 5 millimolarEDTA, 2% dextran (molecular weight from 70 to 200 kilodaltons), 0.05micrograms per milliliter of IgM antibodies to glycophorin A. Also inFIG. 24, AVIBeads are magnetic beads for capturing white blood cells and“Antibodies” refer to antibodies that bind white blood cells.

By analyzing various fractions of the sample after processing stepsusing fluorescence in situ hybridization, the discovery was made thatenriched fetal cells could be detected in the supernatant of the secondwash (step 1, above; shown in the box as “Supernatant W2” in FIG. 24).The enrichment of fetal cells in the Wash 2 supernatant however was notas good as the enrichment of fetal cells collected after filtration(step 3) and their condition was relatively poor.

Fetal cells were isolated from maternal blood cells by centrifuging ablood sample at low speed (such as between 900 and 2000 rpm for 4 to 10min.) and removing the supernatant (Wash 1). A buffer (PBE) was added tothe cell pellet, and the sample was centrifuged again at low speed, atapproximately 1000 rpm for 10 minutes (Wash 2). The supernatant from thesecond centrifugation was analyzed for the presence of nucleated cells.The Wash 2 supernatant was removed from the pellet and put in a fresh 50ml tube. The supernatant was then centrifuged at a speed of 1500 rpm for10 minutes. The supernatant from this pelleting step was removed, andthe cell pellet was resuspended with AVIWash (PBE) to a small volume andPrepacyte Treatment was added in equal parts. Aliquots were put onslides and analyzed using an interphase FISH protocol. The nucleatedfetal cells were seen to be intact. The figure shows the number of cellsrecovered.

Aliquots of sample and other sample fractions at specific steps of theoverall enrichment procedure were also analyzed using FISH, as showndiagrammatically in FIG. 24. However, most of these fractions were notfound to have satisfactory enrichment of fetal nucleated cells. Thus,the most consistent and satisfactory cells are located in the recoveredcells after the filtration step. These cells are can be FISHed orlabeled with antibodies and FISHed.

Example 15 Isolation of Fetal Cells from the Second Wash (W2)Supernatant Maternal Blood Sample

In a representative experiment, a maternal blood sample with agestational age of about 13 weeks was divided into three 10 milliliteraliquots, for enriching fetal cells.

Fetal cells were isolated from maternal blood cells by centrifuging theblood sample aliquots at 1500 rpm (about 470×g) for 6 min., with thebrake set at 900 rpm (approximately 170×g) and removed the supernatant(Wash 1). PBE was added to the cell pellet, and the sample wascentrifuged again at low speed, at 1000 rpm (approximately 209×g, rangeof 500-2200 rpm, approximately 52 to 1000×g) for 10 minutes (range of2-30 minutes) with the brake set at 900 rpm approximately 170×g)(Wash2). The break was released at 900 rpm (approximately 170×g) to minimizethe vibration in the slowing down of the centrifuge rotor. This pointmay exist at different speeds in other centrifuges and could be avoidedby removing the ability of the centrifuge to turn on the break.

The second wash (Wash 2 or W2) supernatants from the secondcentrifugation were analyzed for the presence of nucleated cells. TheWash 2 supernatants were removed from the cell pellets and put in fresh50 ml tubes. The W2 supernatants were then centrifuged at a speed of1500 rpm (approximately 470×g) for 10 minutes. The supernatants fromthis pelleting step was removed, and the cell pellets were resuspendedin 750 microliters of PBE, for W2 supernatants from the 10 millilitersample aliquots, respectively.

The resuspended W2 Supernant cells were processed in different ways. Inone case, an equal volume of the RBC aggregation solution was added tothe resuspended cells. In the other cases, beads coated with CD31Antibody were added in 4×10̂8 and 8×10̂8 quantities to the pelletre-suspension. These samples were allowed to rotate for 0.5 hr (range of5 to 120 minutes) and settle against a magnet for 0.5 hr (range of 5 to120 minutes). After settling, the supernatant was allowed to flowthrough the silicon filter chip. Aliquots were put on slides andanalyzed using an interphase FISH protocol. The nucleated fetal cellswere seen to be intact. The figure shows the number of cells recovered.

Aliquots of sample and other sample fractions at various steps of theoverall enrichment procedure were also analyzed using FISH, as showndiagrammatically in FIG. 24. However, most of these fractions were notfound to have satisfactory enrichment of fetal nucleated cells.

Example 16 Separation of Rare Cells from a Blood Sample with Labeling ofBlood Sample Components During Debulking (Fluorescent Prelabeling ofTarget Cells)

To isolate fetal cells from a maternal blood sample, 10 milliliters ofmaternal blood (gestational age 10-20 weeks is centrifuged at 1500 rpm(approximately 470×g) in a 50 milliliter conical tube for 6 minutes. Thesupernatant is aspirated to remove the supernatant, and then wash couldbe repeated. The supernatant was removed and five additional millilitersof PBE is added to the tube to bring the sample up to the originalvolume.

An equal volume (10 milliliters) of Aviprep (PBS lacking calcium andmagnesium containing: 5 millimolar EDTA, 2% dextran (molecular weightfrom 70 to 200 kilodaltons), 0.05 micrograms per milliliter of IgMantibodies to glycophorin A) is added to the tube. An aliquot ofantibody precoated magnetic beads that bind the antigen CD50 is added tothe tube. To improve the enrichment of fetal cells, it is possible toadd an aliquot of antibody precoated magnetic beads that bind to theantigen CD31 to the tube. Also, it is possible to add other precoatedbeads to increase the removal of WBCs and/or increase the amount ofantibody to glycophorin A to increase removal of RBCs. In addition, itis possible to label the desired cells with a labeled antibody thatbinds to an antigen on the desired cells including but not limited to anantigen such as the transferring receptor (CD71). This labeled antibodycan be conjugated to the fluorescent label (e.g. fluorescein labeledantibody to CD71, Leinco Technologies, St. Louis, Mo.) and added to thetube. The tube is inverted for 30 minutes (range of 5 to 120 minutes) atroom temperature in the dark.

After incubation in a red blood cell sedimenting solution and specificbinding members that bind white blood cells and platelets, the tube ispositioned against a magnet and allowed to sit for 30 minutes (range of5 to 120 minutes) in the dark to allow red blood cells to sediment andwhite blood cells and platelets to be captured. After 30 minutes, thesupernatant (the phase above the aggregated red blood cells) iscollected and place in a new tube. The tube is filled with PBE,inverted, and is centrifuged at 1000 rpm for 10 min to pellet cells. Thesupernatant is removed and the pelleted cells are resuspended in 10 uLsof PBE. The resuspended cells are placed on a microscope slide andallowed to dry. When viewed by fluorescence microscopy, labeled fetalcells are fluorescent and seen to be surface labeled.

A separate aliquot of the cell preparation is fixed and permeabalizedfor FISH using a DNA probe used to test for trisomy. The cells labeledwith the labeled antibody will be cells which include nRBCs and fetalcells. It is possible to reduce the number of contaminating maternalcells based on the presence of the fluorescent surface label.

Example 17 Separation of Rare Cells from a Blood Sample with Labeling ofBlood Sample Components During Separation of Undesirable Components forFurther Enrichment of Rare Cells (Fluorescent Prelabeling of TargetCells)

To isolate fetal cells from a maternal blood sample, 10 milliliters ofmaternal blood (gestational age 10-20 weeks) is dispensed into a 50milliliter conical tube. After washing the blood sample, an equal volume(10 milliliters) of Aviprep (PBS lacking calcium and magnesiumcontaining: 5 millimolar EDTA, 2% dextran (molecular weight from 70 to200 kilodaltons), 0.05 micrograms per milliliter of IgM antibodies toglycophorin A) is added to the tube. An aliquot of magnetic beads thatbind CD50 and an aliquot of magnetic beads that CD31 are also added tothe tube. In addition, a labeled antibody that binds CD71 and that isconjugated to a fluorescent label (e.g. fluorescein labeled antibody toCD71, Leinco Technologies, St. Louis, Mo.) is added to the tube. Thelabel antibody is to an antigen present on the potential target cell(e.g. fetal cell). The tube is inverted for 30 minutes at roomtemperature in the dark.

After incubation in a red blood cell sedimenting solution and specificbinding members that bind white blood cells and platelets, the tube ispositioned against a magnet and allowed to sit for 30 minutes to allowred blood cells to sediment and white blood cells and platelets to becaptured in the dark. After 30 minutes, the supernatant (the phase abovethe red blood cells) is collected and place in a new tube.

The recovered supernatant is then filtered through a filtration chamberhaving dimension of that comprises a filter having 3.2 micron wide slotsthat divides the filtration chamber into an antechamber and apost-filtration subchamber. The antechamber comprises a polycarbonateball suspended from the loading reservoir inlet by four fins that areattached to the sides of the antechamber.

The sample is filtered at a rate of 20 milliliters per hour. After thereservoir is emptied, 3 additional milliliters of PBE is filteredthrough to wash the chamber. Finally, a backwash is performed in whichthe waste outlet is closes, and 5 milliliters of PBE is pumped into thelower post-filtration subchamber through the side outlet. The washsolution is pushed into the post-filtration chamber to dislodge anycells that may have collected or aggregated below the filter. The lowerwaste outlet is opened again, and filtration of the backwash through thefilter is performed. After the backwash, the chamber is rotated and thefiltered sample is collected from the antechamber.

The sample is then analyzed by flow cytometry and the labeled, nucleatedcells are recovered. The cells are cytospun onto a slide and analyzed.

Another possibility is to have the supernatant removed after filtrationand the pelleted cells are resuspended in 10 uLs of PBE. The resuspendedcells are placed on a microscope slide and allowed to dry. When viewedby fluorescence microscopy, labeled nucleated cells are fluorescent andseen to be surface labeled. This would assist to identify potentialfetal cells.

A separate aliquot of the cell preparation is fixed and permeabalizedfor FISH using a DNA probe used to test for trisomy. Fetal cells can bedistinguished from maternal cells by the presence of the fluorescentsurface label nucleated cells.

Example 18 Separation of Rare Cells from a Blood Sample with Labeling ofBlood Sample Components During Separation of Undesirable Components forEnrichment of Rare Cells (Fluorescent Prelabeling of Non-Target Cells)

To isolate fetal cells from a maternal blood sample, 10 milliliters ofmaternal blood (gestational age 10-20 weeks) is dispensed into a 50milliliter conical tube. After washing the blood sample and the sampleis resuspend with PBE to the original volume, an equal volume (10milliliters) of Aviprep (PBS lacking calcium and magnesium containing: 5millimolar EDTA, 2% dextran (molecular weight from 70 to 200kilodaltons), 0.05 micrograms per milliliter of IgM antibodies toglycophorin A) is added to the tube. An aliquot of magnetic beads thatbind CD50 and an aliquot of magnetic beads that CD31 are also added tothe tube. In addition, a labeled antibody that binds a cell surfacemarker (including but not limited to e.g. CD7) and that is conjugated tothe detectable label (including but not limited to e.g. fluoresceinlabeled antibody to CD7, Ancell, Bayport, Minn.) is added to the tube.The labeled antibody is to an antigen that is not or minimally presenton the potential desired cell (e.g. fetal cell), and one example is theantigen CD7 which is expressed less on the desired cells compared toWBCs. The tube is inverted for 30 minutes at room temperature in thedark.

After incubation in a red blood cell sedimenting solution and specificbinding members that bind white blood cells and platelets, the tube ispositioned against a magnet and allowed to sit for 30 minutes in thedark to allow red blood cells to sediment and white blood cells andplatelets to be captured in the dark. After 30 minutes, the supernatant(the phase above the red blood cells) is collected and place in a newtube.

The recovered supernatant is then filtered through a filtration chamberhaving dimension that comprises a filter having 3.2 micron wide slotsthat divides the filtration chamber into an antechamber and apost-filtration subchamber. The sample is filtered at a rate of 20milliliters per hour. After the reservoir is emptied, 3 additionalmilliliters of PBE is filtered through to wash the chamber. Finally, thebottom chamber wash is performed in which the top chamber outlet isclosed, and 5 milliliters of PBE (five cycles of one milliliter PBEfollowed by a volume of air) is pumped into the lower post-filtrationsubchamber through the side outlet. The wash solution is pushed into thepost-filtration chamber to dislodge any cells that may have collected oraggregated below the filter. The lower waste outlet is closed, and thetop chamber inlet is opened. After the wash, the chamber is rotated andthe filtered sample is collected from the antechamber.

The sample is then analyzed by flow cytometry and the unlabeled,nucleated cells are recovered. The cells are cytospun onto a slide andanalyzed.

Another possibility is to have the supernatant removed after filtrationand the pelleted cells are resuspended in 10 uLs of PBE. The resuspendedcells are placed on a microscope slide and allowed to dry. When viewedby fluorescence microscopy, labeled nucleated cells are fluorescent,seen to be surface labeled, and removed from analysis. This would assistto identify potential fetal cells, which would be unlabeled andnucleated.

A separate aliquot of the cell preparation is fixed and permeabalizedfor FISH using a DNA probe used to test for trisomy. Maternal cells canbe distinguished from fetal cells by the presence of the fluorescentsurface label nucleated cells.

Example 19 A Ball Cartridge Configuration May Permit Increased SampleLoading and Increased Yield of Fetal Cells from a Maternal Blood Sample

A cartridge configuration for the filter chips referred to as a ballconfiguration was tested against a standard cartridge configuration forthe ability to filter fetal cells from maternal blood. The ball-likeconfiguration permitted increased sample loading and filtration, whichresulted in increased fetal cell yield. Fetal cells were observed when30 mL of blood was filtered using the ball cartridge configuration. Theconfiguration of the cartridge is based on having a ball-shaped objectin the middle of the top cartridge. The ball redirects the fluid flow tobe more uniform in fluid distribution on the filter chip compared to acartridge configuration without one. The present cartridge configurationhas most of the fluid flow in the direct middle as shown in figure YYand this is compared to a ball cartridge configuration.

To demonstrate the increased fetal cell yield findings blood sample MB7555 (as described) was used as the maternal blood sample. A total of 40mL of MB ∩7555 was aliquoted into four separate 50 mL conical vialslabeled samples 1, 2a, 2b and 2c. Each was washed two times with PBE.Each was spun at 1500 rpm (approximately 470×g) for 6 min and aspiratedto 7.5 mL. Each sample was returned into its original 10 mL volume withPBE. Washed anti-CD50 precoated beads were added to samples 1 through 2cand washed anti-CD31 precoated beads were added to the samples. Eachsample was rotated for 30 minutes. Each sample was uncapped and put intoa Dynal magnet for 30 minutes at room temperature. The supernatant wasrecovered and filtered through a filter chip.

Sample 1 was added to a regular cartridge and samples 2a-2c were addedto a ball cartridge. The captured cells were counted using ahemocytometer. The cells were then spun at 1000 rpm (approximately209×g) for 10 min with brake off at zero. The supernatant was removedand cells were smeared onto the appropriate number of slides and airdried for 1 hr at room temperature.

Fixation Protocol

A portion of each sample was fixed for 15 minutes at room temperature inmethanol (MEOH)/acetic acid (AA) at a ration of 3:1. Each was washed in2×SSC for four minutes at room temperature. Each was dehydrated in 70%,90%, 100% ethanol for four minutes each at room temperature and allowedto air dry. Slides were viewed on a microscope to determine the amountof denaturing time.

XY FISH Protocol

For each slide use: 0.5 uL X Probe and 0.5 uL Y Probe, 7.0 uLHybridization buffer, 2.0 distilled water. The hybridization mixture wasput onto coverslips and the slide was turned upside down and placed onhybridization mixture. Each slide/coverslip was sealed with rubbercement. Slides were denatured at 80 degrees Celsius for an allotted timein Vysis Hybrite machine (Vysis, Downers Grove, Ill.). The slides wereput into a slide rack in a Tupperware container containing damp papertowels and were hybridized overnight in an oven at 37 degrees Celsius.After incubation, the rubber cement and cover slip was removed. Eachslide was singly placed in 0.4×SSC at 70 degrees Celsius for 30 seconds.Each slide was placed in 2×SSC with 0.1% NP40 at room temperature for 1minute. The slides were removed, the backs wiped and allowed to air dry.A drop of Vectashield with DAPI (Vector Laboratories, Burlingame,Calif.) was place on a coverslip, placed on the slide and sealed withnail polish.

Experiment XY FISH Polycarbonate Ball 30 mL Samples MB7557, 13 weeksgestation, Jul. 27, 2004 drawn Operator Jia Xu/Charina Schmitigal DateJul. 27, 2004 AviPrep Sample 1 Sample 2 Procedure and Results Time Drawn9:23 am Treatment None None Type of wash buffer PBE w/ Heparin PBE w/Heparin Speed of the wash in rpms twice at 1500 for 6 minutes Wash stepW1 and W2, aspirated to 7.5 mL Start samples in mls 30 10 AVISolution w/10 U/ml 10 mls each with 1.25 ug of GpA per 10 mls heparin (GpALot#040715001) AVIsolution Specs. Jul. 16, 2004 PGH RBCs per ml 5.60E+095.60E+09 Bead Manufactor AVIVA WBCs per ml 7.20E+06 7.20E+06 Type &AVIBead storage 1.0 mm Neutravidin AVIBeads in buffer Bang's buffer w/0.3% Azide Bead Lot 040707001 for CD31 and 040621001 for CD50 Beads ofCD50/WBC 40 40 μgs of CD50 per 10{circumflex over ( )}9 beads 25 25 Lotof AVIVA CD50 030902004 Beads of CD31 1.00E+09 1.00E+09 μgs of CD31 per10{circumflex over ( )}9 beads 25 25 Rock 30 min at RT in one 50 mlConical tube Magnetic Stand 30 min at RT in Dynal magnet mls recovered  40.5   13.2 Sl Membrane Depletion Chip Manufacturer Samsung Chip NameT18_05 T18_37 Chip Type and Set-Up Polycarbonate Ball, Control 30 mLBlood Speed (ml/hr) 40 20 Fetal Cells  4  0 Number of slides made  3  4

Example 20 Validation of Fetal Cell Enrichment Protocol to Isolate FetalCells from Maternal Peripheral Blood

To isolate fetal cells from a maternal peripheral blood sample, samples(9 milliliters per tube for a total of 27 mls) of maternal peripheralblood (gestational age 10-18 weeks) was dispensed into a three 50milliliter conical tubes. Each tube containing an aliquot of blood wasfilled with PBE and washed once using a single centrifugation of 1000rpms (˜209×g) for 8 minutes. After removing the supernatant from thesample and resuspension of the sample with PBE to the original volume,an equal volume (9 milliliters) of AVIPrep (1×PBS lacking calcium andmagnesium containing: 5 millimolar EDTA, 2% dextran (molecular weightfrom 70 to 200 kilodaltons), 0.15 micrograms of IgM antibody toglycophorin A per milliliter) is added to the tube. An aliquot ofmagnetic beads that bind CD50 (40 beads per white blood cell) and analiquot of magnetic beads that CD31 (3.6×10⁹ beads) were added to eachtube.

After rotation at room temperature for 30 minutes (range of 5 to 120minutes) in a red blood cell aggregating solution and magnetic specificbinding members that bind white blood cells and platelets, the tube waspositioned against a magnet for 30 minutes (range of 5-120 minutes). Thered blood aggregates were allowed to sediment and white blood cells andplatelets were captured by the magnet. The supernatant (the phase abovethe red blood cells) was collected and place in a new tube.

The supernatant was loaded into a filtration chamber containing a filterbiochip (2.8 to 3.2 micron wide slots) that divides the filtrationchamber into an antechamber and a post-filtration subchamber. After thereservoir containing the supernatant was emptied, 3 milliliters of PBE(three cycles of one milliliter PBE followed by a volume of air) wasfiltered through to wash the chamber. Finally, the bottom chamber washwas performed in which the top chamber outlet was closed, and 5milliliters of PBE (five cycles of one milliliter PBE followed by avolume of air) was pumped into the lower post-filtration subchamberthrough the side outlet. The wash solution was pushed into thepost-filtration chamber to dislodge any cells that may have collected oraggregated below the filter. The lower waste outlet was closed, and thetop chamber inlet was opened. The chamber was rotated and the filteredsample containing the enriched cells was collected from the antechamber.The cells were pelleted using a centrifugation step (1000 rpm (˜209×g)for 10 minutes) and the cells were placed onto a slide, fixed,hybridized to chromosome probes using interphase FISH and analyzed forY⁺ cells.

TABLE Amount of male fetal cells isolated from peripheral blood of womencarrying a male fetus. MB # Total Fetal Cells (+ & ?) Gestation 6204 110 wk 6212 2 10 wk 6198 2 10 wk 6250 2 12 wk 6106 2 13 wk 6110 2 13 wk6239 2 14 wk 1163 2 12 wk 6278 3 10 wk 6202 3 11 wk 6258 3 11 wk 6107 314 wk 1155 3 17 wk 6242 4 12 wk 6118 4 15 wk 6210 5 16 wk 6272 10 11 wk6276 12 10 wk 6189 12 17 wk 6117 18 14 wk 6266 2 17 wk

Example 21 Fetal Cells Isolated from Maternal Peripheral Blood andIdentification of Fetal Cells Using Immunocytochemistry or In SituHybridization and Fluorescent In Situ Hybridization

During enrichment of rare fetal cells using methods disclosed herein,the sample and various sample fractions were tested for the presence andabundance of nucleated fetal cells. This is presented schematically inFIG. 24. The figure shows a fetal cell enrichment procedure that beginswith a maternal blood sample (upper left) and ends in a high-qualitypreparation of enriched fetal cells.

It is possible to include an identification step after cell enrichmentand before FISH to identify fetal cells. It is also possible to utilizelaser capture microdissection on labeled cells to obtain a higher purityof fetal cells. This could include using an instrument (e.g. ArcturusPixCell) to catapult the desired cell onto cap. Pulsing the laserthrough the cap to bridge the gap between the cap and desired cell andadheres to the target cell. Lifting the cap removes the target cell(s)now attached to the cap. Biomolecules can then be extracted from thecells using DNA, RNA or protein isolation kits.

One example is the following. The steps of the enrichment procedure,going in sequential order and from upper left to lower right in thefigure, are: 1) washing the blood sample (1 or 2 centrifugations); 2)selectively sedimenting red blood cells and selectively removing whiteblood cells with a Combined Reagent (PBS lacking calcium and magnesiumcontaining: 5 millimolar EDTA, 2% dextran (molecular weight from 70 to200 kilodaltons), 0.125 or 0.15 micrograms per milliliter of IgMantibodies to glycophorin A, and approximately 1-5×10⁹ magnetic beadscoated with a CD50 antibody and approximately 1-4×10⁹ magnetic beadscoated with a CD31 antibody); and 3) filtering the supernatant of step2) through a microfabricated filter, such as the microfabricated filtersdescribed in [Examples 15 and 16]. The sample then is deposited onto amicroscope slide.

The cells are fixed with a fixation agent (100% S.T.F. (Streck, Omaha,Nebr.) for 10 minutes and S.T.F.-0.75% PFA for 4 minutes), washed inwater, and washed in 1×PBS for 6 minutes each. The cells could thenincubated with antibodies to the following antigens: including but notlimited to anti-ε hemoglobin, anti-γ hemoglobin, anti-α-fetoprotein,anti-Epidermal growth factor receptor (EGFR), and anti-c-erbB-2/HER2.Theses antibodies could be detected by primary fluorescent label,secondary antibody, enzymatic labeling or combination of all three.

It is also possible to use probes to the RNA or DNA sequence to identifyRNA or DNA present in fetal cells. The cells could then incubated withnucleic acids (including but not limited to e.g. oligonucleotides,antisense RNA or DNA oligonucleotides, peptide nucleic acids) tohybridize with the following sequences: including but not limited toanti-ε hemoglobin, anti-γ hemoglobin, anti-α-fetoprotein, anti-Epidermalgrowth factor receptor (EGFR), and anti-c-erbB-2/HER2. These sequencescould be detected by primary fluorescent label, secondary antibody,enzymatic labeling or combination of all three.

After the immunohistochemistry or immunocytochemical or in situhybridization reaction, the slides are washed and dehydrated withethanol washes. The slides are denatured by an incubated at 80 degreesCelsius for 1.75 to 5 minutes with fluorescently labeledoligonucleotides (0.5 μl of CEP X and 0.5 μl of CEP Y (Vysis, DownersGrove, Ill.), 2 μl of dH₂O, and 7 μl of CEP buffer solution). Aftermelting the DNA, the slides are incubated at 37 degrees Celsiusovernight. The slides are washed at 70 degrees Celsius for 20-30 secondsin 0.4×SSC and once in 2×SSC and 0.1% Igepal CA-630 for 1 minute. Thenuclei are stained with a nuclear specific fluorescent dye. Ananti-fading reagent (e.g. VectorShield Vector) is add to the cells andthe slide is cover slipped. The cells on the slide are then read usingfluorescent microscopy.

It is then possible to target the labeled cells for lasermicrodissection using the fluorescent label(s) as a cell marker. Thelabeled cell(s) could be targeted for laser microdissection after cellenrichment, after cell labeling step or after interphase FISH procedure.The slide can be transferred to a laser capture device and catapult thedesired cell onto cap, which has been placed over the target area.Pulsing the laser through the cap causes the thermoplastic film to forma thin protrusion that bridges the gap between the cap and desired celland adheres to the target cell. Lifting the cap removes the targetcell(s) now attached to the cap. Biomolecules are extracted from thecells using DNA, RNA or protein isolation kits.

By obtaining a relative pure subpopulation containing the target cellsof interest (e.g. fetal cells), it is now possible to study the cellsdirectly. This would result in the ability to study a further enrichedfetal cell subpopulation using many technologies including but notlimited to mass spec (e.g. protein and SNP analysis), microarray (e.g.chromosome alterations, point or genomic alterations and SNP analysis),and whole genome amplification for analysis of single to few cells (e.g.study point mutations or genomic alterations).

All publications, including patent documents and scientific articles,referred to in this application and the bibliography and attachments areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication were individually incorporatedby reference.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

What is claimed is:
 1. A method of enriching specific types of rarecells (e.g. fetal, cancer, stem, subpopulation of blood, etc.) from abody fluid sample (e.g. blood, ascites, urine, etc), comprising: a)obtaining a sample; b) centrifuging said sample, optionally at a speedof between 10×g and 3000×g and time between 15 seconds and 30 minutes toobtain a sample wash pellet and a supernatant; c) resuspending saidsample wash pellet to obtain a washed sample for rare cell enrichment;d) performing at least one debulking step on said washed sample for rarecell enrichment; e) and performing at least one separation on saidwashed sample for rare cell enrichment to obtain enriched rare cells. 2.The method of claim 1, wherein said debulking step comprises adding tosaid blood sample a solution that selectively sediments red blood cells.3. The method of claim 1, wherein said separation comprises adding atleast one specific binding member that selectively binds white bloodcells and/or other undesired products.
 4. The method of claim 3, furthercomprising adding a reagent to remove other products from blood otherthan red or white blood cells.
 5. The method of claim 4, wherein saiddepletion of other products includes non-normal hematopoietic cells(e.g. cancer cells, epithelial cells, viral infected cells).